Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 aamp;amp;times; 107 and 1 aamp;amp;times; 108 vRNA copies/mL at 3aamp;amp;ndash;10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 aamp;amp;times; 106 and 1 aamp;amp;times; 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies.
Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive Ptight promoter at the 5aamp;amp;prime; end and an antigenomic ribozyme of hepatitis delta virus at the 3aamp;amp;prime; end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection.
The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals.
Zika virus (ZIKV) is an emerging pathogen member of the Flaviviridae family. ZIKV has spread rapidly in the Latin American region, causing hundreds of thousands of cases of ZIKV disease, as well as microcephaly in congenital infections. Detailed studies on the pattern of evolution of ZIKV strains have been extremely important to our understanding of viral survival, fitness, and evasion of the hostaamp;amp;rsquo;s immune system. For these reasons, we performed a comprehensive phylogenetic analysis of ZIKV strains recently isolated in the Americas. The results of these studies revealed evidence of diversification of ZIKV strains circulating in the Latin American region into at least five different genetic clusters. This diversification was also reflected in the different trends in dinucleotide bias and codon usage variation. Amino acid substitutions were found in E and prM proteins of the ZIKV strains isolated in this region, revealing the presence of novel genetic variants circulating in Latin America.
Magnaporthe oryzae, the fungus that causes rice blast, is the most destructive pathogen of rice worldwide. A number of M. oryzae mycoviruses have been identified. These include Magnaporthe oryzae. viruses 1, 2, and 3 (MoV1, MoV2, and MoV3) belonging to the genus, Victorivirus, in the family, Totiviridae; Magnaporthe oryzae. partitivirus 1 (MoPV1) in the family, Partitiviridae; Magnaporthe oryzae. chrysovirus 1 strains A and B (MoCV1-A and MoCV1-B) belonging to cluster II of the family, Chrysoviridae; a mycovirus related to plant viruses of the family, Tombusviridae (Magnaporthe oryzae. virus A); and a (+)ssRNA mycovirus closely related to the ourmia-like viruses (Magnaporthe oryzae. ourmia-like virus 1). Among these, MoCV1-A and MoCV1-B were the first reported mycoviruses that cause hypovirulence traits in their host fungus, such as impaired growth, altered colony morphology, and reduced pigmentation. Recently we reported that, although MoCV1-A infection generally confers hypovirulence to fungi, it is also a driving force behind the development of physiological diversity, including pathogenic races. Another example of modulated pathogenicity caused by mycovirus infection is that of Alternaria alternata chrysovirus 1 (AaCV1), which is closely related to MoCV1-A. AaCV1 exhibits two contrasting effects: Impaired growth of the host fungus while rendering the host hypervirulent to the plant, through increased production of the host-specific AK-toxin. It is inferred that these mycoviruses might be epigenetic factors that cause changes in the pathogenicity of phytopathogenic fungi.
Countries rely on good diagnostic tests and appropriate testing schemes to fight against economically important small ruminant lentivirus (SRLV) infections. We undertook an extensive comparative analysis of seven commercially available serological tests and one in-house real-time PCR (qPCR) detecting genotype A and B strains using a large panel of representative Belgian field samples and samples from experimentally infected sheep and goats. ELISAs generally performed well and detected seroconversion within three weeks post experimental infection. Two enzyme-linked immunosorbent assays (ELISAs) (Elitest and IDscreenaamp;amp;reg; kits) showed the highest sensitivities (aamp;amp;gt;96%) and specificities (aamp;amp;gt;95%) in both species, and their combined use allowed to correctly identify the infection status of all animals. Individual agar gel immunodiffusion (AGIDs) kits lacked sensitivity, but interestingly, the combined use of both kits had a sensitivity and specificity of 100%. qPCRs detected SRLV infection before seroconversion at two weeks post infection and showed a specificity of 100%. Sensitivity however remained suboptimal at 85%. These results allow to propose a faster and cheaper diagnostic testing strategy for Belgium by combining a first ELISA screening, followed by confirmation of positive samples in AGID and/or a second ELISA. Since genotypes A and B strains are predominant in many countries, these results are interesting for other countries implementing SRLV control programs.
Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (aamp;amp;Delta;arrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.
Arthropod vectors control the replication of arboviruses through their innate antiviral immune responses. In particular, the RNA interference (RNAi) pathways are of notable significance for the control of viral infections. Although much has been done to understand the role of RNAi in vector populations, little is known about its importance in non-vector mosquito species. In this study, we investigated the presence of an RNAi response in Toxorhynchites amboinensis, which is a non-blood feeding species proposed as a biological control agent against pest mosquitoes. Using a derived cell line (TRA-171), we demonstrate that these mosquitoes possess a functional RNAi response that is active against a mosquito-borne alphavirus, Semliki Forest virus. As observed in vector mosquito species, small RNAs are produced that target viral sequences. The size and characteristics of these small RNAs indicate that both the siRNA and piRNA pathways are induced in response to infection. Taken together, this data suggests that Tx. amboinensis are able to control viral infections in a similar way to natural arbovirus vector mosquito species. Understanding their ability to manage arboviral infections will be advantageous when assessing these and similar species as biological control agents.
Human Cytomegalovirus (hCMV), which is the prototype member of the aamp;amp;beta;-subfamily of the herpesvirus family, is a pathogen of high clinical relevance in recipients of hematopoietic cell transplantation (HCT). hCMV causes multiple-organ disease and interstitial pneumonia in particular upon infection during the immunocompromised period before hematopoietic reconstitution restores antiviral immunity. Clinical investigation of pathomechanisms and of strategies for an immune intervention aimed at restoring antiviral immunity earlier than by hematopoietic reconstitution are limited in patients to observational studies mainly because of ethical issues including the imperative medical indication for chemotherapy with antivirals. Aimed experimental studies into mechanisms, thus, require animal models that match the human disease as close as possible. Any model for hCMV disease is, however, constrained by the strict host-species specificity of CMVs that prevents the study of hCMV in any animal model including non-human primates. During eons of co-speciation, CMVs each have evolved a set of aamp;amp;ldquo;private genesaamp;amp;rdquo; in adaptation to their specific mammalian host including genes that have no homolog in the CMV virus species of any other host species. With a focus on the mouse model of CD8 T cell-based immunotherapy of CMV disease after experimental HCT and infection with murine CMV (mCMV), we review data in support of the phenomenon of aamp;amp;ldquo;biological convergenceaamp;amp;rdquo; in virus-host adaptation. This includes shared fundamental principles of immune control and immune evasion, which allows us to at least make reasoned predictions from the animal model as an experimental aamp;amp;ldquo;proof of concept.aamp;amp;rdquo; The aim of a model primarily is to define questions to be addressed by clinical investigation for verification, falsification, or modification and the results can then give feedback to refine the experimental model for research from aamp;amp;ldquo;bedside to benchaamp;amp;rdquo;.
The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of a neural chronic inflammation, called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and of a malignant lymphoproliferation, called the adult T-cell leukemia/lymphoma (ATLL). The mechanisms through which the HTLV-1 induces these diseases are still unclear, but they might rely on immune alterations. HAM/TSP is associated with an impaired production of pro-inflammatory cytokines and chemokines, such as IFN-aamp;amp;gamma;, TNF-aamp;amp;alpha;, CXCL9, or CXCL10. ATLL is associated with high levels of IL-10 and TGF-aamp;amp;beta;. These immunosuppressive cytokines could promote a protumoral micro-environment. Moreover, HTLV-1 infection impairs the IFN-I production and signaling, and favors the IL-2, IL-4, and IL-6 expression. This contributes both to immune escape and to infected cells proliferation. Here, we review the landscape of cytokine dysregulations induced by HTLV-1 infection and the role of these cytokines in the HTLV-1-associated diseases progression.
The infection by dengue virus (DENV) of microglia causes cell activation and migration via a mechanism involving viral entry, RNA release, and Toll-like receptor 3 signaling. In this study, we demonstrated that secreted chemotactic factors present in microglial conditioned medium (MCM) facilitated cell motility in the murine BV2 microglial cells. The pharmacological disruption of lipid rafts/caveolae reduced DENV- and ultraviolet (UV)-inactivated MCM-induced microglial cell migration. An antibody-based cytokine/chemokine array showed an increase in macrophage inflammatory protein (MIP)-3aamp;amp;beta; in MCM produced using DENV-infected cells. The pharmacological inhibition of c-Jun N-terminal kinase (JNK) retarded UV-MCM-induced microglial cell migration. These results demonstrate that secreted MIP-3aamp;amp;beta; and its effect on the JNK signaling pathways mediates DENV-induced BV2 microglial cell migration.
Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-aamp;amp;beta; upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.
The rise of multidrug-resistant bacteria has resulted in an increased interest in phage therapy, which historically preceded antibiotic treatment against bacterial infections. To date, there have been no reports of serious adverse events caused by phages. They have been successfully used to cure human diseases in Eastern Europe for many decades. More recently, clinical trials and case reports for a variety of indications have shown promising results. However, major hurdles to the introduction of phage therapy in the Western world are the regulatory and legal frameworks. Present regulations may take a decade or longer to be fulfilled. It is of urgent need to speed up the availability of phage therapy.
Cryphonectria hypovirus 1 (CHV-1) infects the chestnut blight fungus Cryphonectria parasitica and acts as a biological control agent against this harmful tree disease. In this study, we screened the recently characterized C. parasitica population in Eurasian Georgia for the presence of CHV-1. We found 62 CHV-1 infected C. parasitica isolates (9.3%) among a total of 664 isolates sampled in 14 locations across Georgia. The prevalence of CHV-1 at the different locations ranged from 0% in the eastern part of the country to 29% in the western part. Sequencing of two specific regions of the viral genome one each in ORFA and ORFB revealed a unique CHV-1 subtype in Georgia. This subtype has a recombinant pattern combining the ORFA region from the subtype F2 and the ORFB region from subtype D. All 62 viral strains belonged to this Georgian CHV-1 subtype (subtype G). The CHV-1 subtype G strongly reduced the parasitic growth of C. parasitica isolates from Georgia, with a more severe effect on the European genepool compared to the Georgian genepool. The CHV-1 subtype detected in Georgia provides a valuable candidate for biological control applications in the Caucasus region.
Small Ubiquitin-like MOdifier (SUMO) conjugation to proteins has essential roles in several processes including localization, stability, and function of several players implicated in intrinsic and innate immunity. In human, five paralogs of SUMO are known of which three are ubiquitously expressed (SUMO1, 2, and 3). Infection by rhabdoviruses triggers cellular responses through the activation of pattern recognition receptors, which leads to the production and secretion of interferon. This review will focus on the effects of the stable expression of the different SUMO paralogs or Ubc9 depletion on rhabdoviruses-induced interferon production and interferon signaling pathways as well as on the expression and functions of restriction factors conferring the resistance to rhabdoviruses.
The reliable identification and classification of infectious diseases is critical for understanding their biology and controlling their impact. Recent advances in sequencing technology have allowed insight into the remarkable diversity of the virosphere, of which a large component remains undiscovered. For these emerging or undescribed viruses, the process of classifying unknown sequences is heavily reliant on existing nucleotide sequence information in public databases. However, due to the enormous diversity of viruses, and past focus on the most prevalent and impactful virus types, databases are often incomplete. Picobirnaviridae is a dsRNA virus family with broad host and geographic range, but with relatively little sequence information in public databases. The family contains one genus, Picobirnavirus, which may be associated with gastric illness in humans and animals. Little further information is available due in part to difficulties in identification. Here, we investigate diversity both within the genus Picobirnavirus and among other dsRNA virus types using a combined phylogenetic and functional (protein structure homology-modelling) approach. Our results show that diversity within picobirnavirus exceeds that seen between many other dsRNA genera. Furthermore, we find that commonly used practices employed to classify picobirnavirus, such as analysis of short fragments and trimming of sequences, can influence phylogenetic conclusions. The degree of phylogenetic and functional divergence among picobirnavirus sequences in our study suggests an enormous undiscovered diversity, which contributes to the undescribed aamp;amp;ldquo;viral dark matteraamp;amp;rdquo; component of metagenomic studies.
In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.
There is an increasing frequency of reports regarding the persistence of the Ebola virus (EBOV) in Ebola virus disease (EVD) survivors. During the 2014aamp;amp;ndash;2016 West African EVD epidemic, sporadic transmission events resulted in the initiation of new chains of human-to-human transmission. Multiple reports strongly suggest that these re-emergences were linked to persistent EBOV infections and included sexual transmission from EVD survivors. Asymptomatic infection and long-term viral persistence in EVD survivors could result in incidental introductions of the Ebola virus in new geographic regions and raise important national and local public health concerns. Alarmingly, although the persistence of filoviruses and their potential for sexual transmission have been documented since the emergence of such viruses in 1967, there is limited knowledge regarding the events that result in filovirus transmission to, and persistence within, the male reproductive tract. Asymptomatic infection and long-term viral persistence in male EVD survivors could lead to incidental transfer of EBOV to new geographic regions, thereby generating widespread outbreaks that constitute a significant threat to national and global public health. Here, we review filovirus testicular persistence and discuss the current state of knowledge regarding the rates of persistence in male survivors, and mechanisms underlying reproductive tract localization and sexual transmission.
Respiratory viral infections are strongly associated with asthma exacerbations. Rhinovirus is most frequently-detected pathogen; followed by respiratory syncytial virus; metapneumovirus; parainfluenza virus; enterovirus and coronavirus. In addition; viral infection; in combination with genetics; allergen exposure; microbiome and other pathogens; may play a role in asthma development. In particular; asthma development has been linked to wheezing-associated respiratory viral infections in early life. To understand underlying mechanisms of viral-induced airways disease; investigators have studied respiratory viral infections in small animals. This report reviews animal models of human respiratory viral infection employing mice; rats; guinea pigs; hamsters and ferrets. Investigators have modeled asthma exacerbations by infecting mice with allergic airways disease. Asthma development has been modeled by administration of virus to immature animals. Small animal models of respiratory viral infection will identify cell and molecular targets for the treatment of asthma.
Rift Valley fever phlebovirus (RVFV), the causative agent of an emerging zoonotic disease in Africa and Arabia, can infect a variety of species, predominantly ruminants, camelids, and humans. While clinical symptoms are mostly absent in adult ruminants and camelids, RVFV infection may lead to a serious, sometimes fatal disease in humans. Virus transmissions between individuals and between species mainly occur through mosquito bites, but direct or even indirect contact with infectious materials may also result in infection. Although the main reservoir of the virus is not yet identified, small mammals such as rodents and bats may act as amplifying hosts. We therefore inoculated Rousettus aegyptiacus fruit bats that are abundant in northern Africa with the vaccine strain MP-12, in order to elucidate the general competence of this species for virus propagation and transmission. We were able to detect the RVFV genome in the spleen of each of these animals, and re-isolated the virus from the spleen and liver of some animals. Moreover, we were able to identify the Gc RVFV surface antigen in mild subacute multifocal necrotizing hepatic lesions of one bat which was sacrificed 7 days post exposure. These findings demonstrate that Rousettus aegyptiacus fruit bats can propagate RVFV.
Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.
Since emerging in 2012, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has been a global public health threat with a high fatality rate and worldwide distribution. There are no approved vaccines or therapies for MERS until now. Passive immunotherapy with neutralizing monoclonal antibodies (mAbs) is an effective prophylactic and therapeutic reagent against emerging viruses. In this article, we review current advances in neutralizing mAbs against MERS-CoV. The receptor-binding domain (RBD) in the spike protein of MERS-CoV is a major target, and mouse, camel, or human-derived neutralizing mAbs targeting RBD have been developed. A major problem with neutralizing mAb therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mAbs targeting different epitopes. Neutralizing mAbs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against MERS-CoV infection.
Ebola virus is the causative agent of Ebola virus disease in humans. The lethality of Ebola virus infection is about 50%, supporting the urgent need to develop anti-Ebola drugs. Glycoprotein (GP) is the only surface protein of the Ebola virus, which is functionally critical for the virus to attach and enter the host cells, and is a promising target for anti-Ebola virus drug development. In this study, using the recombinant HIV-1/Ebola pseudovirus platform we previously established, we evaluated a small molecule library containing various quinoline compounds for anti-Ebola virus entry inhibitors. Some of the quinoline compounds specifically inhibited the entry of the Ebola virus. Among them, compound SYL1712 was the most potent Ebola virus entry inhibitor with an IC50 of ~1 aamp;amp;mu;M. The binding of SYL1712 to the vial glycoprotein was computationally modeled and was predicted to interact with specific residues of GP. We used the time of the addition assay to show that compound SYL1712 blocks Ebola GP-mediated entry. Finally, consistent with being an Ebola virus entry inhibitor, compound SYL1712 inhibited infectious Ebola virus replication in tissue culture under biosafety level 4 containment, with an IC50 of 2 aamp;amp;mu;M. In conclusion, we identified several related molecules with a diaryl-quinoline scaffold as potential anti-EBOV entry inhibitors, which can be further optimized for anti-Ebola drug development.
Finding a cure for HIV is challenging because the virus is able to integrate itself into the host cell genome and establish a silent state, called latency, allowing it to evade antiviral drugs and the immune system. Various aamp;amp;ldquo;shock and killaamp;amp;rdquo; strategies are being explored in attempts to eliminate latent HIV reservoirs. The goal of these approaches is to reactivate latent viruses (aamp;amp;ldquo;shockaamp;amp;rdquo;), thereby exposing them to clearance by viral cytopathic effects or immune-mediated responses (aamp;amp;ldquo;killaamp;amp;rdquo;). To date, there has been limited clinical success using these methods. In this review, we highlight various functions of the HIV accessory protein Nef and discuss their double-edged effects that may contribute to the limited effectiveness of current aamp;amp;ldquo;shock and killaamp;amp;rdquo; methods to eradicate latent HIV reservoirs in treated individuals.
Arctic marine ecosystems are currently undergoing rapid changes in temperature and light availability. Picophytoplankton, such as Micromonas polaris, are predicted to benefit from such changes. However, little is known about how these environmental changes affect the viruses that exert a strong mortality pressure on these small but omnipresent algae. Here we report on one-step infection experiments, combined with measurements of host physiology and viability, with 2 strains of M. polaris and the virus MpoV-45T under 3 light intensities (5, 60 and 160 aamp;amp;mu;mol quanta maamp;amp;minus;2 saamp;amp;minus;1), 2 light period regimes (16:8 and 24:0 h light:dark cycle) and 2 temperatures (3 and 7 aamp;amp;deg;C). Our results show that low light intensity (16:8 h light:dark) delayed the decline in photosynthetic efficiency and cell lysis, while decreasing burst size by 46%. In contrast, continuous light (24:0 h light:dark) shortened the latent period by 5 h for all light intensities, and even increased the maximum virus production rate and burst size under low light (by 157 and 69%, respectively). Higher temperature (7 aamp;amp;deg;C vs 3 aamp;amp;deg;C) led to earlier cell lysis and increased burst size (by 19%), except for the low light conditions. These findings demonstrate the ecological importance of light in combination with temperature as a controlling factor for Arctic phytoplankton host and virus dynamics seasonally, even more so in the light of global warming.
Multiple viruses with zoonotic potential have been isolated from bats globally. Here we describe the isolation and characterization of a novel paramyxovirus, Alston virus (AlsPV), isolated from urine collected from an Australian pteropid bat colony in Alstonville, New South Wales. Characterization of AlsPV by whole-genome sequencing and analyzing antigenic relatedness revealed it is a rubulavirus that is closely related to parainfluenza virus 5 (PIV5). Intranasal exposure of mice to AlsPV resulted in no clinical signs of disease, although viral RNA was detected in the olfactory bulbs of two mice at 21 days post exposure. Oronasal challenge of ferrets resulted in subclinical upper respiratory tract infection, viral shedding in respiratory secretions, and detection of viral antigen in the olfactory bulb of the brain. These results imply that AlsPV may be similar to PIV5 in its ability to infect multiple mammalian host species. This isolation of a novel paramyxovirus with the potential to transmit from bats to other mammalian species reinforces the importance of continued surveillance of bats as a source of emerging viruses.
Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death. At present, no effective treatment for EV71 is available. We reviewed here various mouse models for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains. To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71 clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate viral entry in cell culture. However, in vivo infection with human SCARB2 receptor transgenic mice appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors, immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed in models deficient in various arms of innate or humoral immunity. These models are being actively used for antiviral research.
An atypical porcine pestivirus (APPV) causing congenital tremor type A-II in piglets was identified in China in 2016. An increased number of cases of APPV have been reported in various countries all over the world since 2015. This study aimed to develop an effective subunit vaccine against APPV based on the E2 protein, which is the main immunogenicity protein of APPV. In this study, E2 protein was successfully expressed by the baculovirus expression system. E2 protein was confirmed by Western blot assay, which showed that E2 protein possesses N-linked glycosylation sites. The immunogenicity of E2 subunit vaccine was evaluated in mice. The E2 protein emulsified with ISA 201VG adjuvant induced significantly higher levels of APPV-specific antibodies and elicited stronger lymphocyte proliferative responses and higher interleukin-10 secretion than those of the E2 protein emulsified with IMS 1313VG adjuvant. This observation indicates that the E2 subunit vaccine induces a Th2-type immune response. Our results showed that E2 protein can be developed as a safe and effective subunit vaccine for the control of APPV infection.
The St. Lawrence hydrographic system includes freshwater, brackish, and marine habitats, and is the largest waterway in North America by volume. The food-webs in these habitats are ultimately dependent on phytoplankton. Viral lysis is believed to be responsible for a major part of phytoplankton mortality. To better understand their role, we characterized the diversity and distribution of two viral taxa infecting phytoplankton: the picornaviruses and phycodnaviruses. Our study focused on the estuary transition zone, which is an important nursery for invertebrates and fishes. Both viral taxa were investigated by PCR amplification of conserved molecular markers and next-generation sequencing at six sites, ranging from freshwater to marine. Our results revealed few shared viral phylotypes between saltwater and freshwater sites. Salinity appeared to be the primary determinant of viral community composition. Moreover, our analysis indicated that the viruses identified in this region of the St. Lawrence diverge from classified viruses and homologous published environmental virotypes. These results suggest that DNA and RNA viruses infecting phytoplankton are likely active in the estuary transition zone, and that this region harbors its own unique viral assemblages.
At the end of my doctoral studies, in 1988, I published a review article on the major steps of transcription and translation during the mammalian reovirus multiplication cycle, a topic that still fascinates me 30 years later. It is in the nature of scientific research to generate further questioning as new knowledge emerges. Our understanding of these fascinating viruses thus remains incomplete but it seemed appropriate at this moment to look back and reflect on our progress and most important questions that still puzzle us. It is also essential of being careful about concepts that seem so well established, but could still be better validated using new approaches. I hope that the few reflections presented here will stimulate discussions and maybe attract new investigators into the field of reovirus research. Many other aspects of the viral multiplication cycle would merit our attention. However, I will essentially limit my discussion to these central aspects of the viral cycle that are transcription of viral genes and their phenotypic expression through the host cell translational machinery. The objective here is not to review every aspect but to put more emphasis on important progress and challenges in the field.
Murine herpesvirus-68 (MHV-68) productively infects mouse lungs, exhibiting a complex pathology characteristic of both acute viral infections and chronic respiratory diseases. We sought to discover proteins differentially expressed in bronchoalveolar lavage (BAL) from mice infected with MHV-68. Mice were infected intranasally with MHV-68. After nine days, as the lytic phase of infection resolved, differential BAL proteins were identified by two-dimensional (2D) electrophoresis and mass spectrometry. Of 23 unique proteins, acute phase proteins, vitamin A transport, and oxidative stress response factors Pdx6 and EC-SOD (Sod3) were enriched. Correspondingly, iNOS2 was induced in lung tissue by seven days post-infection. Oxidative stress was partly a direct result of MHV-68 infection, as reactive oxygen species (ROS) were induced in cultured murine NIH3T3 fibroblasts and human lung A549 cells infected with MHV-68. Finally, mice infected with a recombinant MHV-68 co-expressing inflammatory cytokine murine interleukin 6 (IL6) showed exacerbated oxidative stress and soluble type I collagen characteristic of tissue recovery. Thus, oxidative stress appears to be a salient feature of MHV-68 pathogenesis, in part caused by lytic replication of the virus and IL6. Proteins and small molecules in lung oxidative stress networks therefore may provide new therapeutic targets to ameliorate respiratory virus infections.
Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identified RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent.
Siphoviridae of the lactococcal 936 group are the most commonly encountered bacteriophages in the dairy processing environment. The 936 group phages possess a discrete baseplate at the tip of their tailaamp;amp;mdash;a complex harbouring the Receptor Binding Protein (RBP) which is responsible for host recognition and attachment. The baseplate-encoding region is highly conserved amongst 936 phages, with 112 of 115 publicly available phages exhibiting complete synteny. Here, we detail the three exceptions (Phi4.2, Phi4R15L, and Phi4R16L), which differ from this genomic architecture in possessing an apparent second RBP-encoding gene upstream of the aamp;amp;ldquo;classicalaamp;amp;rdquo; rbp gene. The newly identified RBP possesses an elongated neck region relative to currently defined 936 phage RBPs and is genetically distinct from defined 936 group RBPs. Through detailed characterisation of the representative phage Phi4.2 using a wide range of complementary techniques, we demonstrated that the above-mentioned three phages possess a complex and atypical baseplate structure. Furthermore, the presence of both RBPs in the tail tip of the mature virion was confirmed, while the anticipated host-binding capabilities of both proteins were also verified.
The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 aamp;amp;Aring; resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display.
Classical insect-specific flaviviruses (cISFs) have been widely detected in different countries in the last decades. Here, we characterize the near full-length genomes of two cISFs detected in mosquitoes collected in the city of Macapaamp;amp;aacute;, state of Amapaamp;amp;aacute;, Amazon region of Brazil. A total of 105 pools of female mosquitos were analyzed by next-generation sequencing (NGS). Comparative genomics and phylogenetic analysis identified three strains of cell fusing agent virus (CFAV) and two of Culex flavivirus (CxFV). All sequences were obtained from pools of Culex sp., except for one sequence of CFAV detected in a pool of Aedes aegypti. Both CxFV strains are phylogenetically related to a strain isolated in 2012 in the Southeast region of Brazil. The CFAV strains are the first of this species to be identified in Brazil and one of them is highly divergent from other strains of CFAV that have been detected worldwide. In conclusion, CFAV and CxFV, circulate in mosquitoes in Brazil. One strain of CFAV is highly divergent from others previously described, suggesting that a novel strain of CFAV is present in this region.
The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera Beauveria, Metarhizium, Lecanicillium, Purpureocillium, Isaria, and Paecilomyces were screened for the presence of dsRNA elements and 12 Spanish B. bassiana isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised Beauveria bassiana victorivirus 1 (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual B. bassiana isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly Ceratitis capitata showed no direct correlation between the presence of any particular mycovirus and the virulence of the B. bassiana isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future.
RNA silencing is a conserved surveillance mechanism against invading viruses in plants, which involves the production of virus-derived small interfering RNAs (vsiRNAs) that play essential roles in the silencing of viral RNAs and/or specific host transcripts. However, how vsiRNAs function to target viral and/or host transcripts is poorly studied, especially in maize (Zea mays L.). In this study, a degradome library constructed from Sugarcane mosaic virus (SCMV)-inoculated maize plants was analyzed to identify the cleavage sites in viral and host transcripts mainly produced by vsiRNAs. The results showed that 42 maize transcripts were possibly cleaved by vsiRNAs, among which several were involved in chloroplast functions and in biotic and abiotic stresses. In addition, more than 3000 cleavage sites possibly produced by vsiRNAs were identified in positive-strand RNAs of SCMV, while there were only four cleavage sites in the negative-strand RNAs. To determine the roles of vsiRNAs in targeting viral RNAs, six vsiRNAs were expressed in maize protoplast based on artificial microRNAs (amiRNAs), of which four could efficiently inhibit the accumulations of SCMV RNAs. These results provide new insights into the genetic manipulation of maize with resistance against virus infection by using amiRNA as a more predictable and useful approach.
In 2017, Hadidi et al. edited a voluminous monograph entitled aamp;amp;ldquo;Viroids and Satellitesaamp;amp;rdquo;, in which each known viroid and viroid-like satellite species was described in detail from many perspectives by more than 100 experts from 24 countries. In its 700+ pages, the book is a much needed detailed and reliable compendium of a subject, which, undoubtedly, is still little known by many potential readers. Because most users of the book may be expected to be practical plant pathologists, it appears essential that the book contain, in addition to the detailed viroid and satellite descriptions, one chapter, in which the basic molecular biology of viroids and satellites is described.
Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112aamp;amp;ndash;119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.
To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3aamp;amp;ndash;4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.
Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.
Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.
Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1aamp;amp;ndash;4 aamp;amp;mu;M significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC50 values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 aamp;amp;mu;M. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-aamp;amp;alpha;, IFN-aamp;amp;beta;, IL-1aamp;amp;alpha;, IL-6, IL-8 and TNF-aamp;amp;alpha;) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine.
Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration.
Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000aamp;amp;ndash;300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.
The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 aamp;amp;deg;C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.
H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.
We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 aamp;amp;deg;C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.
Rift Valley fever virus (RVFV) is an emerging pathogen capable of causing severe disease in livestock and humans and can be transmitted by multiple routes including aerosol exposure. Several animal models have been developed to gain insight into the pathogenesis associated with aerosolized RVFV infection, but work with these models is restricted to high containment biosafety level (BSL) laboratories limiting their use for antiviral and vaccine development studies. Here, we report on a new RVFV inhalation infection model in STAT2 KO hamsters exposed to aerosolized MP-12 vaccine virus by nose-only inhalation that enables a more accurate delivery and measurement of exposure dose. RVFV was detected in hepatic and other tissues 4aamp;amp;ndash;5 days after challenge, consistent with virus-induced lesions in the liver, spleen and lung. Furthermore, assessment of blood chemistry and hematological parameters revealed alterations in several liver disease markers and white blood cell parameters. Our results indicate that STAT2 KO hamsters develop a disease course that shares features of disease observed in human cases and in other animal models of RVFV aerosol exposure, supporting the use of this BSL-2 infection model for countermeasure development efforts.
Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-aamp;amp;beta;-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.
Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus.
One of the crucial steps during trials for Zika and other vaccines is to recruit participants and to understand how participantsaamp;amp;rsquo; attitudes and sociodemographic characteristics affect willingness to participate (WTP). This study was conducted to assess WTP, its explanatory variables, and the impact of financial compensation on WTP in Indonesia. A health facility-based cross-sectional study was conducted in eleven regencies in the Aceh and West Sumatra provinces of Indonesia. Participants were recruited via a convenience sampling method and were interviewed. The associations between explanatory variables and WTP were assessed using a two-step logistic regression analysis. A total of 1,102 parents were approached, and of these 956 (86.8%) completed the interview and were included in analysis. Of those, 144 (15.1%) were willing to participate in a Zika vaccine trial without a financial compensation. In the multivariate analysis, WTP was tied to an age of more than 50 years old, compared to 20aamp;amp;ndash;29 years (odds ratio (OR): 5.0; 95% confidence interval (CI): 2.37aamp;amp;ndash;10.53), to being female (OR: 2.20; 95% CI: 1.11aamp;amp;ndash;4.37), and to having heard about Zika (OR: 2.41; 95% CI: 1.59aamp;amp;ndash;3.65). Participantsaamp;amp;rsquo; WTP increased gradually with higher financial compensation. The rate of WTP increased to 62.3% at the highest offer (US$ 350.4), and those who were still unwilling to participate (37.7%) had a poorer attitude towards childhood vaccination. This study highlights that pre-existing knowledge about Zika and attitudes towards childhood vaccination are important in determining community members being willing to participate in a vaccine trial. Financial incentives are still an important factor to enhance participant recruitment during a vaccine trial.
Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants.
Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities.
Hepatitis C virus (HCV)-specific T cell responses are closely linked to the clinical course of infection. While T cell responses in self-limiting infection are typically broad and multi-specific, they display several distinct features of functional impairment in the chronic phase. Moreover, HCV readily adapts to immune pressure by developing escape mutations within epitopes targeted by T cells. Much of our current knowledge on HCV-specific T cell responses has been gathered under the assumption that this might eventually pave the way for a therapeutic vaccine. However, with the development of highly efficient direct acting antivirals (DAAs), there is less interest in the development of a therapeutic vaccine for HCV and the scope of T cell research has shifted. Indeed, the possibility to rapidly eradicate an antigen that has persisted over years or decades, and has led to T cell exhaustion and dysfunction, provides the unique opportunity to study potential T cell recovery after antigen cessation in a human in vivo setting. Findings from such studies not only improve our basic understanding of T cell immunity but may also advance immunotherapeutic approaches in cancer or chronic hepatitis B and D infection. Moreover, in order to edge closer to the WHO goal of HCV elimination by 2030, a prophylactic vaccine is clearly required. Thus, in this review, we will summarize our current knowledge on HCV-specific T cell responses and also provide an outlook on the open questions that require answers in this field.
Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.
The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viralaamp;amp;ndash;host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism.
The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies.
The influenza A virus (IAV) genome consists of eight single-stranded RNA segments. Each segment is associated with a protein complex, with the 3aamp;amp;prime; and 5aamp;amp;prime; ends bound to the RNA-dependent RNA polymerase (RdRp) and the remainder associated with the viral nucleoprotein. During transcription of viral mRNA, this ribonucleoprotein complex steals short, 5aamp;amp;prime;-capped transcripts produced by the cellular DNA dependent RNA polymerase II (RNAPII) and uses them to prime transcription of viral mRNA. Here, we review the current knowledge on the process of IAV cap-snatching and suggest a requirement for RNAPII promoter-proximal pausing for efficient IAV mRNA transcription.
The mechanisms that drive formation of the HIV capsid, first as an immature particle and then as a mature protein shell, remain incompletely understood. Recent discoveries of positively-charged rings in the immature and mature protein hexamer subunits that comprise them and their binding to the cellular metabolite inositol hexakisphosphate (IP6) have stimulated exciting new hypotheses. In this paper, we discuss how data from multiple structural and biochemical approaches are revealing potential roles for IP6 in the HIV-1 replication cycle from assembly to uncoating.
Human bocaviruses (HBoVs) 1aamp;amp;ndash;4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1aamp;amp;ndash;4 DNAs in the blood and stool samples, and of HBoV1aamp;amp;ndash;4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virusaamp;amp;mdash;most frequently norovirus or rotavirusaamp;amp;mdash;was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are.
Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 aamp;amp;micro;M of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.
Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of aamp;amp;ldquo;consensus-degenerate hybrid oligonucleotide primersaamp;amp;rdquo; (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.
Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Schaamp;amp;ouml;ntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Schaamp;amp;ouml;ntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field.
The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8aamp;amp;alpha;+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-aamp;amp;gamma; mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillainaamp;amp;minus;Barraamp;amp;eacute; syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mLaamp;amp;minus;1 and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV.
Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.
Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.
The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection.
The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development.
Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.
Hymenoscyphus albidus is a native fungus in Europe where it behaves as a harmless decomposer of leaves of common ash. Its close relative Hymenoscyphus fraxineus was introduced into Europe from Asia and currently threatens ash (Fraxinus sp.) stands all across the continent causing ash dieback. H. fraxineus isolates from Europe were previously shown to harbor a mycovirus named Hymenoscyphus fraxineus Mitovirus 1 (HfMV1). In the present study, we describe a conspecific mycovirus that we detected in H. albidus. HfMV1 was consistently identified in H. albidus isolates (mean prevalence: 49.3%) which were collected in the sampling areas before the arrival of ash dieback. HfMV1 strains in both fungal hosts contain a single ORF of identical length (717 AA) for which a mean pairwise identity of 94.5% was revealed. The occurrence of a conspecific mitovirus in H. albidus and H. fraxineus is most likely the result of parallel virus evolution in the two fungal hosts. HfMV1 sequences from H. albidus showed a higher nucleotide diversity and a higher number of mutations compared to those from H. fraxineus, probably due to a bottleneck caused by the introduction of H. fraxineus in Europe. Our data also points to multiple interspecific virus transfers from H. albidus to H. fraxineus, which could have contributed to the intraspecific virus diversity found in H. fraxineus.
The spread of viral pathogens both between and within hosts is inherently a spatial process. While the spatial aspects of viral spread at the epidemiological level have been increasingly well characterized, the spatial aspects of viral spread within infected hosts are still understudied. Here, with a focus on influenza A viruses (IAVs), we first review experimental studies that have shed light on the mechanisms and spatial dynamics of viral spread within hosts. These studies provide strong empirical evidence for highly localized IAV spread within hosts. Since mathematical and computational within-host models have been increasingly used to gain a quantitative understanding of observed viral dynamic patterns, we then review the (relatively few) computational modeling studies that have shed light on possible factors that structure the dynamics of spatial within-host IAV spread. These factors include the dispersal distance of virions, the localization of the immune response, and heterogeneity in host cell phenotypes across the respiratory tract. While informative, we find in these studies a striking absence of theoretical expectations of how spatial dynamics may impact the dynamics of viral populations. To mitigate this, we turn to the extensive ecological and evolutionary literature on range expansions to provide informed theoretical expectations. We find that factors such as the type of density dependence, the frequency of long-distance dispersal, specific life history characteristics, and the extent of spatial heterogeneity are critical factors affecting the speed of population spread and the genetic composition of spatially expanding populations. For each factor that we identified in the theoretical literature, we draw parallels to its analog in viral populations. We end by discussing current knowledge gaps related to the spatial component of within-host IAV spread and the potential for within-host spatial considerations to inform the development of disease control strategies.
The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.
Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naaamp;amp;iuml;ve HIV-1 infected individuals in northern Thailand during 2001aamp;amp;ndash;2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.
We present the recently isolated virus vB_BthP-Goe4 infecting Bacillus thuringiensis HD1. Morphological investigation via transmission electron microscopy revealed key characteristics of the genus Phi29virus, but with an elongated head resulting in larger virion particles of approximately 50 nm width and 120 nm height. Genome sequencing and analysis resulted in a linear phage chromosome of approximately 26 kb, harbouring 40 protein-encoding genes and a packaging RNA. Sequence comparison confirmed the relation to the Phi29virus genus and genomes of other related strains. A global average nucleotide identity analysis of all identified aamp;amp;phi;29-like viruses revealed the formation of several new groups previously not observed. The largest group includes Goe4 and may significantly expand the genus Phi29virus (Salasvirus) or the Picovirinae subfamily.
Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amapaamp;amp;aacute; (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV (n = 8) and by CHIKV/ZIKV (n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.
Phage-derived depolymerases directed against bacterial capsules are showing therapeutic promise in various animal models of infection. However, individual animal model studies are often constrained by use of highly specific protocols, such that results may not generalize to even slight modifications. Here we explore the robustness of depolymerase therapies shown to succeed in a previous study of mice. Treatment success rates were reduced by treatment delay, more so for some enzymes than others: K1- and K5 capsule-degrading enzymes retained partial efficacy on delay, while K30 depolymerase did not. Phage were superior to enzymes under delayed treatment only for K1. Route of administration (intramuscular versus intraperitoneal) mattered for success of K1E, possibly for K1F, not for K1H depolymerase. Significantly, K1 capsule-degrading enzymes proved highly successful when using immune-suppressed, leukopenic mice, even with delayed treatment. Evolution of bacteria resistant to K1-degrading enzymes did not thwart therapeutic success in leukopenic mice, likely because resistant bacteria were avirulent. In combination with previous studies these results continue to support the efficacy of depolymerases as antibacterial agents in vivo, but system-specific details are becoming evident.
Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.
Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.
The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.
RNA silencing (or RNA interference, RNAi) plays direct roles in plant host defenses against viruses. Viruses encode suppressors of RNAi (VSRs) to counteract host antiviral defenses. The generation of transgenic plants expressing VSRs facilitates the understanding of the mechanisms of VSR-mediated interference with the endogenous silencing pathway. However, studying VSRs independent of other viral components simplifies the complex roles of VSRs during natural viral infection. While suppression of transgene silencing by the VSR 2b protein encoded by cucumber mosaic virus (CMV) requires 2b-small RNA (sRNA) binding activity, suppression of host antiviral defenses requires the binding activity of both sRNAs and AGOs proteins. This study, aimed to understand the functions of 2b in the context of CMV infection; thus, we performed genome-wide analyses of differential DNA methylation regions among wild-type CMV-infected, CMVaamp;amp;Delta;2b-infected, and 2b-transgenic Arabidopsis plants. These analyses, together with transcriptome sequencing and RT-qPCR analyses, show that while the majority of induced genes in 2b-transgenic plants were involved in extensive metabolic pathways, CMV-infection 2b-dependent induced genes were enriched in plant immunity pathways, including salicylic acid (SA) signaling. Together with infection with CMV mutants that expressed the 2b functional domains of sRNA or AGO binding, our data demonstrate that CMV-accelerated SA signaling depends on 2b-sRNA binding activity which is also responsible for virulence.
A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.
The virulent phage ST32 that infects the Escherichia coli strain ST130 was isolated from a wastewater sample in China and analyzed. Morphological observations showed that phage ST32 belongs to the Myoviridae family, as it has an icosahedral capsid and long contractile tail. Host range analysis showed that it exhibits a broad range of hosts including non-pathogenic and pathogenic E. coli strains. Interestingly, phage ST32 had a much larger burst size when amplified at 20 aamp;amp;deg;C as compared to 30 aamp;amp;deg;C or 37 aamp;amp;deg;C. Its double-stranded DNA genome was sequenced and found to contain 53,092 bp with a GC content of 44.14%. Seventy-nine open reading frames (ORFs) were identified and annotated as well as a tRNA-Arg. Only nineteen ORFs were assigned putative functions. A phylogenetic tree using the large terminase subunit revealed a close relatedness with four unclassified Myoviridae phages. A comparative genomic analysis of these phages showed that the Enterobacteria phage phiEcoM-GJ1 is the closest relative to ST32 and shares the same new branch in the phylogenetic tree. Still, these two phages share only 47 of 79 ORFs with more than 90% identity. Phage ST32 has unique characteristics that make it a potential biological control agent under specific conditions.
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus for which there is no vaccine or cure. This viral infection, once acquired, is life-long, residing latently in hematopoietic cells. However, latently infected individuals with weakened immune systems often undergo HCMV reactivation, which can cause serious complications in immunosuppressed and immunocompromised patients. Current anti-viral therapies target late stages of viral replication, and are often met with therapeutic resistance, necessitating the development of novel therapeutics. In this current study, we identified a naturally-occurring flavonoid compound, deguelin, which inhibits HCMV lytic replication. Our findings reveal that nanomolar concentrations of deguelin significantly suppress the production of the infectious virus. Further, we show that deguelin inhibits the lytic cycle during the phase of the replication cycle consistent with early (E) gene and protein expression. Importantly, our data reveal that deguelin inhibits replication of a ganciclovir-resistant strain of HCMV. Together, our findings identify a novel, naturally occurring compound that may prove useful in the treatment of HCMV replication.
Recent developments in defining the role of the lentiviral envelope glycoprotein (Env) cytoplasmic tail (CT) in Env trafficking and incorporation into virus particles have advanced our understanding of viral replication and transmission. To stimulate additional progress in this field, the two-day International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail, co-organized by Eric Freed and James Hoxie, was held at the National Cancer Institute in Frederick, MD (26aamp;amp;ndash;27 April 2018). The meeting served to bring together experts focused on the role of gp41 in HIV replication and to discuss the emerging mechanisms of CT-dependent trafficking, Env conformation and structure, host protein interaction, incorporation, and viral transmission. The conference was organized around the following three main hot topics in gp41 research: the role of host factors in CT-dependent Env incorporation, Env structure, and CT-mediated trafficking and transmission. This review highlights important topics and the advances in gp41 research that were discussed during the conference.
Composed of a naked circular non-protein-coding genomic RNA, counting only a few hundred nucleotides, viroidsaamp;amp;mdash;the smallest infectious agents known so faraamp;amp;mdash;are able to replicate and move systemically in herbaceous and woody host plants, which concomitantly may develop specific diseases or remain symptomless. Several viroids have been reported to naturally infect pome and stone fruit trees, showing symptoms on leaves, fruits and/or bark. However, Kochaamp;amp;rsquo;s postulates required for establishing on firm grounds the viroid etiology of these diseases, have not been met in all instances. Here, pome and stone fruit tree diseases, conclusively proven to be caused by viroids, are reviewed, and the need to pay closer attention to fulfilling Kochaamp;amp;rsquo;s postulates is emphasized.
Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.
Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.
Varicella-zoster virus (VZV) is a pathogenic human herpes virus that causes varicella (chickenpox) as a primary infection, following which it becomes latent in peripheral ganglia. Decades later, the virus may reactivate either spontaneously or after a number of triggering factors to cause herpes zoster (shingles). Varicella and its complications are more severe in the immunosuppressed. The most frequent and important complication of VZV reactivation is postherpetic neuralgia, the cause of which is unknown and for which treatment is usually ineffective. Reactivation of VZV may also cause a wide variety of neurological syndromes, the most significant of which is a vasculitis, which is treated with corticosteroids and the antiviral drug acyclovir. Other VZV reactivation complications include an encephalitis, segmental motor weakness and myelopathy, cranial neuropathies, Guillainaamp;amp;ndash;Barraamp;amp;eacute; syndrome, enteric features, and zoster sine herpete, in which the viral reactivation occurs in the absence of the characteristic dermatomally distributed vesicular rash of herpes zoster. There has also been a recent association of VZV with giant cell arteritis and this interesting finding needs further corroboration. Vaccination is now available for the prevention of both varicella in children and herpes zoster in older individuals.
Polymers are generally considered thermal insulators because the amorphous arrangement of the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies reveal that individual chains of polymers with oriented structures could have high thermal conductivity, because such stretched polymeric chains effectively conduct phonons through polymeric covalent bonds. Previously, we have found that the liquid crystalline assembly composed of one of the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity even though the assembly is based on non-covalent bonds. Despite such potential applicability of biopolymeric assemblies as thermal conductive materials, stability against heating has rarely been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 aamp;amp;deg;C) treatment. The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability against heating processes. Our results provide insight into the future use of biomolecular assemblies for reliable thermal conductive materials.
The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.
Venezuelan equine encephalitis virus (VEEV) is an arbovirus that is associated with robust inflammation that contributes to neurodegenerative phenotypes. In addition to triggering central nervous system (CNS) inflammation, VEEV will also induce mitochondrial dysfunction, resulting in increased cellular apoptosis. In this study, we utilize the TC-83 strain of VEEV to determine the role of mitochondrial oxidative stress in mediating inflammation elicited by murine brain microglial cells. Using an in vitro model, we show that murine microglia are susceptible to TC-83 infection, and that these cells undergo mitochondrial stress as the result of infection. We also indicate that bystander microglia contribute more significantly to the overall inflammatory load than directly infected microglia. Use of a mitochondrial targeted antioxidant, mitoquinone mesylate, greatly reduced the pro-inflammatory cytokines released by both direct infected and bystander microglia. Our data suggest that release of interleukin-1aamp;amp;beta;, a key instigator of neuroinflammation during VEEV infection, may be the direct result of accumulating mitochondrial stress. This data improves our understanding inflammation elicited by murine microglia and will aid in the development of more accurate in vitro and in vivo murine model of VEEV-induced neuroinflammation.
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.
Hendra virus (HeV) and Nipah virus (NiV) are among a group of emerging bat-borne paramyxoviruses that have crossed their species-barrier several times by infecting several hosts with a high fatality rate in human beings. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation in different hosts is lacking. A study of codon usage patterns in henipaviruses may provide some fruitful insight into their evolutionary processes of synonymous codon usage and host-adapted evolution. Here, we performed a systematic evolutionary and codon usage bias analysis of henipaviruses. We found a low codon usage bias in the coding sequences of henipaviruses and that natural selection, mutation pressure, and nucleotide compositions shapes the codon usage patterns of henipaviruses, with natural selection being more important than the others. Also, henipaviruses showed the highest level of adaptation to bats of the genus Pteropus in the codon adaptation index (CAI), relative to the codon de-optimization index (RCDI), and similarity index (SiD) analyses. Furthermore, a comparison to recently identified henipa-like viruses indicated a high tRNA adaptation index of henipaviruses for human beings, mainly due to F, G and L proteins. Consequently, the study concedes the substantial emergence of henipaviruses in human beings, particularly when paired with frequent exposure to direct/indirect bat excretions.
Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytesaamp;amp;rsquo; nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (Aaamp;amp;ndash;H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virusaamp;amp;rsquo; viability. Such findings may have key implications for designing antivirals to target these areas.
The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.