|Journal of General Virology current issue|
The relationship between Epstein-Barr virus (EBV) and the germinal centre (GC) of the asymptomatic host remains an enigma. The occasional appearance of EBV-positive germinal centres in some patients, particularly those with a history of immunosuppression, suggests that EBV numbers in the GC are subject to immune control. The relationship, if any, between lymphoid hyperplasia with EBV-positive germinal centres and subsequent or concurrent lymphomagenesis remains to be clarified. As far as the development of EBV-associated Hodgkin's lymphoma is concerned, the suppression of virus replication, mediated by LMP1 on the one hand, and the loss of B-cell receptor signalling on the other, appears to be an important pathogenic mechanism. A further important emerging concept is that alterations in the microenvironment of the EBV-infected B-cell may be important for lymphomagenesis.
Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3nndash;/nndash; mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3nndash;/nndash; mice. In contrast, MyD88nndash;/nndash; mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.
Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-bbeta; cytokines.
Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. Here, we report a recombinant PR8 influenza virus with the RSV fusion (F) protein epitopes of the subgroup A gene inserted into the influenza virus non-structural (NS) gene (rFlu/RSV/F) that was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F was also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line and CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific haemagglutination inhibition antibody responses against the PR8 influenza virus and RSV neutralization test proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited T helper type 1-dominant cytokine profiles against the RSV strain A2 virus. Taken together, our findings suggested that rFlu/RSV/F was immunogenic in vivo and warrants further development as a promising candidate vaccine.
Virologic surveillance is a critical component of measles management. One of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles is yet to be eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very active in China, providing a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a timespan of nearly two decades. Phylogenetic analysis performed on the 450 nt coding sequence for the C-terminal 150 amino acids of the nucleoprotein (N-450), fusion (F) gene and haemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence of selective pressure for the H protein was found. The substitution rates ranged from 0.75x10nndash;3 substitutions sitenndash;1 yearnndash;1 for H to 1.65x10nndash;3 substitutions sitenndash;1 yearnndash;1 for N-450. The time of most recent common ancestor (TMRCA) for genotype H1 was estimated as approximately 1985 (95 % highest probability density, 1979nndash;1989). Finally, the overall diversity of measles sequences from China decreased from 2005 to 2012, coincident with a substantial decrease in measles cases. The results suggest that detailed evolutionary analyses should facilitate the documentation of eventual measles elimination in China. Moreover, the molecular approaches used in this study can be applied in other countries approaching measles elimination.
Cholesterol is a critical component of the hepatitis C virus (HCV) life cycle, as demonstrated by its accumulation within infected hepatocytes and lipoviral particles. To cope with excess cholesterol, hepatic enzymes ACAT1 and ACAT2 produce cholesteryl esters (CEs), which are destined for storage in lipid droplets or for secretion as apolipoproteins. Here we demonstrate in vitro that cholesterol accumulation following HCV infection induces upregulation of the ACAT genes and increases CE synthesis. Analysis of human liver biopsy tissue showed increased ACAT2 mRNA expression in liver infected with HCV genotype 3, compared with genotype 1. Inhibiting cholesterol esterification using the potent ACAT inhibitor TMP-153 significantly reduced production of infectious virus, but did not inhibit virus RNA replication. Density gradient analysis showed that TMP-153 treatment caused a significant increase in lipoviral particle density, suggesting reduced lipidation. These data suggest that cholesterol accumulation following HCV infection stimulates the production of CE, a major component of lipoviral particles. Inhibition of CE synthesis reduces HCV particle density and infectivity, suggesting that CEs are required for optimal infection of hepatocytes.
To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus find its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface were not receptor determinants and even hampered efficient virusnndash;receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells, but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (2,6-aalpha;-linked over 2,3-aalpha;-linked) notably reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin inhibited both treated and untreated viruses. In desialylated cells, however, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrated that FECV had a sialic acid binding capacity, which was partially masked by virus-associated sialic acids, and that attachment to sialylated compounds could facilitate enterocyte infections. However, sialic acid binding was not a prerequisite for the initiation of infection and virusnndash;receptor engagement was even more efficient after desialylation of cells, indicating that FECV requires sialidases for efficient enterocyte infections.
White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20nndash;29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.
While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.
Human cardioviruses or Saffold viruses (SAFVs) of the family Picornaviridae are newly emerging viruses whose genetic and phenotypic diversity are poorly understood. We report here the full genome sequence of 11 SAFV genotypes from Pakistan and Afghanistan, along with a re-evaluation of their genetic diversity and recombination. We detected 88 SAFV from stool samples of 943 acute flaccid paralysis cases using reverse transcriptase-PCR targeting the 5' untranslated region (UTR). Further characterization based on complete VP1 analysis revealed 71 SAFVs belonging to 11 genotypes, including three previously unidentified genotypes. SAFV showed high genetic diversity and recombination based on phylogenetic, pairwise distance distributions and recombination mapping analyses performed herein. Phylogenies based on non-structural and UTRs were highly incongruent indicating frequent recombination events among SAFVs. We improved the SAFV genotyping classification criteria by determining new VP1 thresholds based on the principles used for the classification of enteroviruses. For genotype assignment, we propose a threshold of 23 and 10 % divergence for VP1 nucleotide and amino acid sequences, respectively. Other members of the species Theilovirus, such as Thera virus and Theilerrrsquo;s murine encephalomyelitis virus, are difficult to classify in the same species as SAFV, because they are genetically distinct from SAFV, with 41nndash;56 % aa pairwise distances. The new genetic information obtained in this study will improve our understanding of the evolution and classification of SAFV.
Here, we report the isolation and functional characterization of mAbs against two murine norovirus (MNV) strains, MNV-1 and WU20, which were isolated following oral infection of mice. The mAbs were screened for reactivity against the respective homologous and heterologous MNV strain by ELISA. Selected mAbs were of IgA, IgG1, IgG2a or IgG2b isotype and showed a range of Western blot reactivities from non-binding to strong binding, suggesting recognition of conformational and linear epitopes. Some of the anti-MNV-1 antibodies neutralized both MNV-1 and WU20 infections in culture and in mice, but none of the anti-WU20 mAbs neutralized either virus. The non-neutralizing anti-MNV-1 IgG2b antibody 5C4.10 was mapped to the S domain of the MNV-1 capsid, whilst the epitopes of the neutralizing anti-MNV-1 IgA antibodies 2D3.7 and 4F9.4 were mapped to the P domain. Generation of neutralization escape viruses showed that two mutations (V339I and D348E) in the C'D' loop of the MNV-1 P domain mediated escape from mAb 2D3.7 and 4F9.4 neutralization. These findings broaden the known neutralizing epitopes of MNV to the main surface-exposed loops of the P domain. In addition, the current panel of antibodies provides valuable reagents for studying norovirus biology and development of diagnostic tools.
The genus Flavivirus includes some of the most important human viral pathogens, and its members are found in all parts of the populated world. The temporal origin of diversification of the genus has long been debated due to the inherent problems with dating deep RNA virus evolution. A generally accepted hypothesis suggests that Flavivirus emerged within the last 10 000 years. However, it has been argued that the tick-borne Powassan flavivirus was introduced into North America some time between the opening and closing of the Beringian land bridge that connected Asia and North America 15 000nndash;11 000 years ago, indicating an even older origin for Flavivirus. To determine the temporal origin of Flavivirus, we performed Bayesian relaxed molecular clock dating on a dataset with high coverage of the presently available Flavivirus diversity by combining tip date calibrations and internal node calibration, based on the Powassan virus and Beringian land bridge biogeographical event. Our analysis suggested that Flavivirus originated ~85 000 (64 000nndash;110 000) or 120 000 (87 000nndash;159 000) years ago, depending on the circumscription of the genus. This is significantly older than estimated previously. In light of our results, we propose that it is likely that modern humans came in contact with several members of the genus Flavivirus much earlier than suggested previously, and that it is possible that the spread of several flaviviruses coincided with, and was facilitated by, the migration and population expansion of modern humans out of Africa.
We demonstrated previously that immunization with a DNA vaccine expressing the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection through a poorly neutralizing antibody response. Here, we further investigated the role of the IgG subclass in this antibody-dependent protection using cytokine co-immunization and cytokine-deficient mice. A significant difference in IgG2a/c but not IgG1 was observed between mice that survived or died following a lethal challenge. Correspondingly, the IgG2a/c response and protection increased in IL-4-deficient mice but decreased in IFN--deficient mice, highlighting the importance of IgG2a/c. In addition, the restoration of protection and E-specific IgG2a/c production in IFN--deficient mice by a T helper (Th) type 1-biased intramuscular immunization suggested that IgG2a/c but not IFN- was the major component for protection. The failure of protection against a direct intracranial challenge indicated that IgG2a/c-mediated protection was restricted to outside the central nervous system. Consistent with this conclusion, passive transfer of E-specific antisera conferred protection only pre-exposure to JEV. Therefore, our data provided evidence that the IgG subclass plays an important role in protection against JEV, particular in poorly neutralizing E-specific antibodies, and Th1-biased IgG2a/c confers better protection than Th2-biased IgG1 against JEV.
The molecular basis for the increased resistance of astrocytes to a non-neuropathogenic strain of West Nile virus (WNV), WNV-MAD78, compared with the neuropathogenic strain WNV-NY remains unclear. Here, we demonstrated that the reduced susceptibility of astrocytes to WNV-MAD78 is due to a combination of both cellular activities as well as viral determinants. Analyses of the viral particle indicated that astrocyte-derived WNV-MAD78 particles were less infectious than those of WNV-NY. Additionally, inhibition of cellular furin-like proteases increased WNV-MAD78 infectious particle production in astrocytes, suggesting that high levels of furin-like protease activity within these cells acted in a cell- and strain-specific manner to inhibit WNV-MAD78 replication. Moreover, analysis of recombinant viruses indicated that the structural proteins of WNV-MAD78 were responsible for decreased particle infectivity and the corresponding reduction in infectious particle production compared with WNV-NY. Thus, the composition of the WNV virion was also a major determinant for viral fitness within astrocytes and may contribute to WNV propagation within the central nervous system. Whether the WNV-MAD78 structural genes reduce virus replication and particle infectivity through the same mechanism as the cellular furin-like protease activity or whether these two determinants function through distinct pathways remains to be determined.
Low-level hepatitis C virus (HCV) RNA may persist in PBMCs after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved a sustained virological response (SVR) after pegylated IFN and ribavirin therapy were analysed 52nndash;66 months after the SVR. HCV RNA was detected in PBMCs from 18 patients (47.4 %), and PBMCs in two patients stained positive for non-structural protein 3 (NS3). Quantification of various cytokine and chemokine transcripts in PBMCs revealed that levels of IL-6, IL-8, IL-12, TNF-aalpha; and macrophage inflammatory protein 1bbeta; were significantly higher in HCV-positive patients than in HCV-negative individuals. In conclusion, persistence of HCV RNA in PBMCs of patients with a SVR appears to be associated with immune activation.
Coxsackievirus B3 (CVB3) infection can trigger myocarditis and can ultimately lead to dilated cardiomyopathy. It is known that CVB3-induced T-cell infiltration into cardiac tissues is one of the pathological factors causing cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T-cell infiltration by two types of CVB3: the H3 WT strain and the YYFF attenuated strain. T-cell activation was confirmed by changes in the distribution of lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 could infect and activate T-cells in vivo and in vitro, and activated T-cells were detected in CVB3-infected mouse hearts. LFA-1 expressed on the surface of these T-cells had been activated through the cAMP/Rap1 pathway. Recombinant lentiviruses expressing VP2 of CVB3 could also induce LFA-1 activation via an increase in cAMP, whilst VP2 of YYFF did not. These results indicated that CVB3 infection increased cAMP levels and then activated Rap1 in T-cells. In particular, VP2, among the CVB3 proteins, might be critical for this activation. This VP2nndash;cAMPnndash;Rap1nndash;LFA-1 axis could be a potential therapeutic target for treating CVB3-induced myocarditis.
Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.
Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel, and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibodies. A widely used preclinical cottontail rabbit papillomavirus (CRPV)/rabbit model has been a surrogate model for high-risk human papillomavirus (HPV) infections. New Zealand white rabbits, either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix), were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were ~10-fold lower than those from the corresponding serum samples, but background noise was lower in tear samples. The isotypes of antibodies in tears were predominantly IgA and IgG. These findings showed clearly that tears could be a surrogate for serum samples for monitoring antibody responses. As collecting tears causes no discomfort and poses no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiological studies as well as for use in clinical practice.
Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here, we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of the nuclear factor NF-B and apoptosis that contributes to virulence, has a Bcl-2-like fold, and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated, and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerization. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we described a previously uncharacterized modification of the VACV protein N1 that provided a new layer of complexity to the biology of this virulence factor, and provided another example of the intricate interplay between poxviruses and the host ubiquitin system.
The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CAnndash;NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50 % of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CAnndash;NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.
Ancient endogenous retroviruses (ERVs), designated endogenous avian retrovirus (EAVs), are present in all Gallus spp. including the chicken, and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously named the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. Here, we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the Gallus gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity with an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs formed three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared with other EAVs or ASLVs, suggesting that these ERVs represented exogenous retroviruses that evolved and integrated into the germline over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggested that they were more ancient than the other EAVs and may have diverged through mutations that accumulated post-integration. Finally, we showed that the ART-CH elements were chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.
The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus.
Nitric oxide (NO) is an important defensive signal in plants but its effects on virus infection are not well understood. Administration of NO-releasing compounds immediately before inoculation of tobacco leaves with potato virus X and tobacco mosaic virus decreased the accumulation of virus, indicating that NO can induce resistance rapidly. Resistance induction was inhibited by co-administration with an NO-scavenging compound or when experiments were done in transgenic tobacco plants expressing increased alternative respiratory pathway capacity due to constitutive expression of the plant mitochondrial enzyme, alternative oxidase (AOX). These results indicate that NO, which inhibits electron transport chain activity, is triggering defensive signalling by inducing changes in mitochondrial reactive oxygen species levels that are in turn regulated by AOX. Experiments using nahG-transgenic plants, which cannot accumulate the defensive plant hormone salicylic acid (SA) showed that NO rapidly induces resistance to virus infection independently of SA. However, this initial state of resistance may be transient. Subsequently, by 5 days post-treatment, NO had caused an increase in pathogenesis-related protein 1 (PR1) expression (a proxy for increased SA biosynthesis), which correlated with a longer-term state of resistance to virus infection. The induction by NO of PR1 accumulation was modified in AOX-transgenic plants. This indicates that the influence of NO on defensive gene expression is in part mediated through its effects on mitochondria.
Wheat dwarf virus (WDV) is a DNA virus belonging to the genus Mastrevirus of the family Geminiviridae. In this study, we report that the Rep protein encoded by WDV is a RNA silencing supressor as determined by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene. The Rep protein was shown to inhibit both local and systemic RNA silencing of the GFP gene as well as the spread of systemic GFP RNA silencing signals. Gel mobility shift assays showed that the Rep protein binds 21 nt and 24 nt small interfering RNA (siRNA) duplexes and single-stranded (ss)-siRNA. To our knowledge, this is the first identification of an RNA silencing suppressor encoded by mastreviruses. Furthermore, deletion mutagenesis indicates that both the N- and C-terminal regions of the Rep protein are not critical for silencing suppression and self-interaction, but the N terminus of Rep is necessary for its pathogenicity.
Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.
|Journal of General Virology Publish Ahead of Print|
Aphid lethal paralysis virus (ALPV; Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new strain of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has a single-stranded RNA genome of 9,940 nucleotides. Based on phylogenetic analysis, ALPV-AP is closely related to ALPV-AM, an ALPV strain isolated from honeybees, Apis mellifera in Spain and Brookings, South Dakota, USA. The distinct evolutionary branches suggest the existence of two lineages of the ALPV virus. One consists of ALPV-AP and ALPV-AM while all other strains of ALPV group into the other lineage. The similarity of ALPV-AP and ALPV-AM is up to 88% at the RNA level, compared to 78-79% between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM is 98-99% for both ORF 1 and ORF2, while only 85-87% for ORF1 and 91-92% for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE products and cDNA clones of the virus genome revealed sequence variation in the 5rrsquo; untranslated regions (5rrsquo;UTR) and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicate that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and propose that ALPV isolates be classified as two separate viral species.
In the past decade there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated "Nhumirim virus"; NHUV) (Pauvolid-Correa et al., 2014) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential, 3rrsquo; UTR sequence homology with alternative flaviviruses, and evaluated the virusrrsquo;s capacity to suppress replication of representative Culex spp. vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines such as C6/36, C710, and Culex quinquefasciatus cells, were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3rrsquo; UTR indicate NHUV to be most similar to viruses within the yellow fever serogroup, Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. Interestingly, NHUV was found to share the fewest conserved sequence elements when compared to traditional insect-specific flaviviruses. This suggests that, despite being apparently insect-specific, this virus likely diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in significant reduction in viral production of West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Japanese encephalitis virus (JEV). The inhibitory effect was most effective against WNV and SLEV with over a million-fold and 10,000-fold reduction in peak titers, respectively. These data indicate the potential modulatory effect of flaviviral mosquito co-infections in the field and serve to identify a potential target for blocking mosquito infection with medically important flaviviruses.
Vaccination is the best measure to prevent influenza pandemics. Here we studied the protective effect against heterologous influenza viruses, including A/reassortant/NYMC X-179A (pH1N1), A/chicken/Henan/12/2004 (H5N1), A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89xA/PR/8/34 (A/Guizhou-X) (H3N2), in mice first vaccinated with a DNA vaccine of hemagglutinin (HA) or neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous virus. We showed that PR8 HA or NA vaccination both protected mice against a lethal dose of the homologous virus; PR8 HA or NA vaccination and then PR8 infection in mice respectively offered poor or excellent protection against a second, heterologous influenza virus challenge. In addition,before the second heterologous influenza infection, the highest antibody level against the NP, M1 and M2 was found in PR8 NA DNA vaccinated and PR8 infected group. The level of induced cellular immunity against NP and M1 showed a trend consistent with that seen in antibody levels. However,PR8HA+NA and then PR8 infection resulted in limited protection against heterologous influenza virus challenges. Results of the present study demonstrated that infection of the homologous influenza virus in mice already immunized with a NA vaccine could provide excellent protection against subsequent infection of a heterologous influenza virus. These findings suggest that NA, a major antigen of influenza virus, could be an important candidate antigen for universal influenza vaccines.
A major research priority for HIV eradication is the elucidation of the events involved in HIV reservoir establishment and persistence. Cell-to-cell transmission of HIV represents an important area of study as it allows for the infection of cell types which are not easily infected by HIV, leading to the establishment of long-lived viral reservoirs. This phenomenon enables HIV to escape elimination by the immune system. This process may also enable HIV to escape suppressive effects of anti-retroviral drugs. During cell-to-cell transmission of HIV, a dynamic series of events ensues at the virological synapse which promote viral dissemination. Cell-to-cell transmission involves various types of cells of the immune system and this mode of transmission has been shown to have an important role in sexual and mother-to-child transmissions of HIV and spread of HIV within the central nervous system and gut-associated lymphoid tissues. There is also evidence that cell-to-cell transmission of HIV occurs between thymocytes and renal tubular cells. Herein, following a brief review of the processes involved at the virological synapse (VS), evidence supporting the role for cell-to-cell transmission of HIV in the maintenance of the HIV reservoir will be highlighted. Therapeutic considerations and future directions for this area of research will be also be discussed.
Classical swine fever virus (CSFV) has a tropism for vascular endothelial cells and immune system cells. The process and release of proinflammatory cytokines, including IL-1bbeta; and IL-18, is one of the fundamental reactions of the innate immune response to viral infection. In this study, we investigated the production of IL-1bbeta; from macrophages following CSFV infection. Our results showed that IL-1bbeta; was upregulated after CSFV infection through activating caspase-1. Subsequent studies demonstrated that reactive oxygen species (ROS) may not be involved in CSFV mediated IL-1bbeta; release. Recently, researchers indicate a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induces IL-1bbeta; secretion and also discovered that p7 protein is a short-lived protein degraded by the proteasome. Together, our observations provide an insight into the mechanism of CSFV-induced inflammatory response.
Viral suppressors of RNA silencing (VSRs) are critical for the success of virus infection and efficient accumulation of the virus progeny. The chrysanthemum virus B p12 protein acts as a transcription factor (TF) to regulate cell size and proliferation favorable for the virus infection. Here we show that the p12 protein suppresses RNA silencing and is able to complement a VSR-deficient unrelated virus. Moreover, p12 counter-silencing activity can be uncoupled from its function as a TF in the nucleus. The altered p12 protein, which lacks nuclear localization signal and is not imported into the nucleus, was able to suppress RNA silencing as efficiently as the native protein. These data reveal new aspects of p12 functioning and identify a novel role for this viral zinc-finger transcription factor. The results provide a general insight into one of the activities of the p12 protein, which appears to possess more than one function.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into CHIKV genome and subgenomic (sg) RNA, as well as into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5rrsquo;-end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 x g. Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated RTCs. The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3rrsquo;dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the CHIKV RTC and mode of action studies on compounds with anti-CHIKV activity.
The 2009 pandemic H1N1 influenza A virus spread across the globe and caused the first influenza pandemic of the 21st century. Many of the molecular factors that contributed to the airborne transmission of this pandemic virus have been determined; however, the direct-contact transmission of this virus remains poorly understood. In this study, we report that a combination of two mutations (N159D and Q226R) in the hemagglutinin (HA) protein of the representative 2009 H1N1 influenza virus A/California/04/2009 (CA04) caused a switch in receptor binding preference from the aalpha;2,6-sialoglycan to the aalpha;2,3-sialoglycan receptor and decreased the binding intensities for both glycans. In conjunction with a significantly decreased replication efficiency in the nasal epithelium, this limited human receptor-binding affinity resulted in inefficient direct-contact transmission of CA04 between guinea pigs. Our findings highlight the role of the HA gene in the transmission of the influenza virus.
Interactions between the viral surface glycoprotein hemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in the HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in the HA genes of two 2009 pandemic H1N1 isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at position 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published 2009 pandemic H1N1 (H1N1pdm09) consensus sequence, the P186 genotype was more readily detected in primary infected and contact naiiuml;ve pigs when inoculated with a heterogeneous mixed stock of Ca/09. By reverse genetics we engineered Ca/09 and Mx/09 genomes by introducing the Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either Ca/09 or Mx/09 backbones resulted in higher and prolonged viral shedding in naiiuml;ve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occur within the receptor binding pocket and the Sb antigenic site, they did not affect serologic cross-reactivity. Relative increases of P186 in publically available sequences from swine H1N1pdm09 viruses supports the experimental data indicating this amino acid substitution confers an advantage in swine.
Rats are known as reservoirs and vectors for several zoonotic pathogens. However, information on the viruses shed by urban wild rats that could pose zoonotic risk to human health is scare. Here, intestinal contents from 20 wild Norway rats (Rattus norvegicus) collected in the city of Berlin, Germany, were subjected to metagenomic analysis of viral nucleic acids. The determined faecal viromes of rats consisted of a variety of known and unknown viruses and were highly variable among the individuals. Members of the families Parvoviridae and Picobirnaviridae represented the most abundant species. Novel picorna-, boca-, sapo- and stool-associated circular ssDNA viruses (SCV) were identified, which showed only low sequence identities to known representatives of the corresponding taxa. In addition, noro- and rotaviruses were detected as potential zoonotic gastroenteritis viruses. However, partial genome sequence analyses indicated that the norovirus was closely related to the recently identified rat norovirus and the rotavirus B was closely related to the rat rotavirus strain IDIR; both viruses clustered separately from respective human virus strains in phylogenetic trees. In contrast, the rotavirus A sequences showed high identities to human and animal strains. Analysis of the nearly complete genome of this virus revealed the known genotypes G3, P and N2 for three of the genome segments, whereas the remaining eight genome segments represented the novel genotypes I20-R11-C11-M10-A22-T14-E18-H13. In conclusion, the results indicate a high heterogeneity of enteric viruses present in urban wild rats; their ability to be transmitted to humans remains to be assessed in future.
During a hospital-based diarrheal disease study conducted in Ho Chi Minh City, Vietnam from 2009nndash;2010, we identified four symptomatic children infected with G26P rotavirus, an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P strain, the whole genome of a representative example was characterized, revealing a novel mixed genotype constellation of G26-P-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine rotaviruses (VP1-3, NSP2-5). We propose that this G26P strain was a result from a pig-human zoonotic transmission, followed by a limited onward transmission train in humans. The identification of such strains has potential implications for vaccine efficacy in Southeast Asia and outlines the utility of whole genome sequencing for studying rotavirus genetic diversity and zoonotic potential during disease surveillance.
The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary-cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcription-polymerase chain reaction (RT-PCR). The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes is mainly attributed to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occur without sequence variation.
The major envelope fusion protein F of budded virus (BV) of baculoviruses consists of two disulfide-linked subunits, an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine (C) in F2 and there are fifteen cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein was analyzed by site-directed mutagenesis. Results indicate that in a functional F protein an inter-subunit disulfide-bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide-bond was formed between C108 and C232, but rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant (TCEP) and free-thiol inhibitors (AMS and DTNB) on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. These data suggest that free thiol/disulfide isomerization was not likely to play roles in viral entry and infectivity.
Human immunodeficiency virus type 2 (HIV-2) infects about 2 million people worldwide. HIV-2 has fewer treatment options than HIV-1, yet may evolve drug resistance more quickly. We have analyzed several novel drugs for anti-HIV-2 activity. It was observed that 5-azacytidine, clofarabine, gemcitabine and resveratrol have potent anti-HIV-2 activity. The EC50 values for 5-azacytidine, clofarabine and resveratrol were found to be significantly lower with HIV-2 compared to that of HIV-1. A time-of-addition assay was used to analyze the ability of these drugs to interfere with HIV-2 replication. Reverse transcription was the likely target for antiretroviral activity. Taken together, several novel drugs have been discovered to have activity against HIV-2. Based upon their known activities, these drugs may elicit enhanced HIV-2 mutagenesis and therefore be useful for inducing HIV-2 lethal mutagenesis. In addition, the data are consistent with HIV-2 reverse transcriptase (RT) being more sensitive than HIV-1 RT to dNTP pool alterations.
Based on the hypothesis that interactions between virions and serum components may influence the outcome of Dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from protein E of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and alpha 2-macroglobulin (aalpha;2M) as novel viral interactors. After confirming the specific binding of both SAP and aalpha;2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species is actually the receptor-activated form of aalpha;2M (aalpha;2M*), that aalpha;2M* can bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4 ggt; DENV1 ~ DENV2 ggt; DENV3, and that this interaction exhibits a strong avidity effect when multivalent binding is favored (KD 8 x10-8 M for DIIIE2). We also show that aalpha;2M* binds to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibits an ED50 of 2.9 x10-8 M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factors interaction network during human DENV infection.
Previous studies have shown that hepatitis C virus (HCV) enters human hepatic cells through interaction with a series of cellular receptors, followed by clathrin-mediated, pH-dependent endocytosis. Here, we investigated the mechanisms of HCV entry into multiple HCV-permissive human hepatocyte-derived cells using trans-complemented HCV particles (HCVtcp). Knockdown of CD81 and claudin-1, or treatment with bafilomycin A1, reduced infection in Huh-7 and Huh7.5.1 cells, suggesting that HCV enters both cell types via receptor-mediated, pH-dependent endocytosis. Interestingly, knockdown of the clathrin heavy chain or dynamin-2, as well as expression of the dominant-negative form of dynamin-2, reduced infection of Huh-7 cells with HCVtcp, whereas infectious entry of HCVtcp into Huh7.5.1 cells was not impaired. Infection of Huh7.5.1 cells with culture-derived HCV (HCVcc) via a clathrin-independent pathway was also observed. Knockdown of caveolin-1, ADP-ribosylation factor 6 (Arf6), flotillin, p21-activated kinase 1 (PAK1) and the PAK1 effector C-terminal binding protein 1 of E1A had no inhibitory effects on HCVtcp infection into Huh7.5.1 cells, thus suggesting that the infectious entry pathway of HCV into Huh7.5.1 cells is not caveolae-mediated, or Arf6- and flotillin-mediated endocytosis and macropinocytosis, but rather may occur via an undefined endocytic pathway. Further analysis revealed that HCV entry is clathrin- and dynamin-dependent in ORL8c and HepCD81/miR122 cells, but productive entry of HCV is clathrin- and dynamin-independent in Hep3B/miR122 cells. Collectively, these data indicate that HCV enters different target cells through different entry routes.
We used stable isotope labelling of amino acids in cell culture and high throughput quantitative mass spectrometry to analyse the protein composition of highly purified wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy including one virus which had been used in a clinical trail. We found that the viral protein abundance and composition was consistent across all types of virus examined except for the virus lacking protein V which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique makes a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus like particles. The raw data have been deposited at ProteomeXchange, identifier PXD001120.
We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a sub-genomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real time. GFP expression in transfected BHK-21 cells reached a maximum approximately 8 hours post-transfection, at which time change in morphology of the cells was consistent with virus-induced cytopathic effect (cpe). However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose-dependent. The 3Dpol aptamers appeared to be more effective than 2llsquo;-C-methylcytidine. Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.
Vaccinia virus (VACV) is a large dsDNA virus encoding about 200 proteins, several of which inhibit apoptosis. Here a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) within and outwith virus infection is presented. VACVs strains engineered to lack each gene separately still block apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy we inserted each gene separately into a VACV strain (vv811) that lacks all these anti-apoptotic proteins and that induces apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved PARP-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain WR compared to derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase 3 during infection by vv811 strains expressing these proteins. These results illustrate that vv811 is a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 is most potent.
Wild freshwater eel populations have dramatically declined in recent last decades in Europe and America, partially through the impact of several factors including the widespread of infectious diseases. The Anguillid rhabdoviruses Eel Virus European X (EVEX) and Eel Virus American (EVA) play potentially a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of Anguillid rhabdoviruses by analyzing sequences from the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of 57 viral strains collected from 7 countries over 40 years using Maximum likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow to clearly distinguish two monophyletic groups, European and American. Results of nucleotide substitution rates per site per year indicate that the P gene is subjected to evolve more rapidly. The nucleotide diversity observed is low (2-3 %) for the three genes, with a significantly higher variability within the P gene which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein could be a potential molecular marker suitable for characterization of distinct genotypes within Anguillid rhabdoviruses. This study provides to our knowledge the first molecular characterization of EVA, brings new insights on the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.
We describe here the metagenomics-derived feline enteric virome in the feces of 25 cats from a single shelter in California. More than 90% of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae, and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses and the first genome of a cyclovirus in a feline were characterized. The RNA dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggests that such animals experience robust viral exposures. This study also increases our understanding of the viral diversity in cats facilitating future evaluation of their pathogenic and zoonotic potentials.
Monoclonal antibodies (MAbs) constitute an important biological tool for influenza hemagglutinin (HA) epitope mapping through generation of escape mutants. The latter could provide insights into immune evasion mechanisms and such knowledge may benefit the future development of vaccines. Several influenza A(H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Herein, we produced MAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using MAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including 3 that were previously described (G172E, N173D and K256E) and 8 novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T, G478E). Among the 6 HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), 3 (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I, K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in 3 different Bris07 escape mutants, appear as an immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.
We isolated and characterized a novel virulent bacteriophage IME-EFm1 specifically infecting multiple-drug resistant Enterococcus faecium. IME-EFm1 is morphologically similar to the family Siphoviridae. It was capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 plaque-forming units (PFU)/cell. These biological characteristics suggested that IME-EFm1 hold the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 is a 42597bp in length, linear, terminally non-redundant double-stranded DNA, with a G+C content of 35.2%. The termini of the phage genome were determined with next generation sequencing data and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited low similarity with other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponds to 90.7% of the genome. 70 putative open reading frames were deduced, and of these, 28 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-beta-lactamase gene was detected in the genome sequence. The identification of antibiotic resistance gene emphasizes the necessity of complete genome sequencing of a phage to ensure it free of any undesirable genes.
The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analyzed using a collection of sixteen European swine H1N1 influenza viruses sampled in 1979-1981 in Germany, the Netherlands, Belgium, Italy and France as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggest a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicate times to the most recent common ancestors of 34-46 years (before 2008) depending on the RNA segment and the method of tree inference.
The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses were detected in man and various animal species, but not in reptiles yet. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (ggt;33,000nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the hemagglutinin-neuraminidase of paramyxoviruses. Analysis of the genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity of the Nidovirales.nnbsp;
Bone marrow stromal antigen 2 (BST-2) also known as Tetherin) is an interferon-inducible gene that functions to block the release of a range of nascent enveloped virions from infected host cells. However, the role of BST-2 in viral pathogenesis remains poorly understood. BST-2 plays a multifaceted role in innate immunity-as it hinders retroviral infection and possibly promotes infection with some rhabdo and orthomyxo viruses. This paradoxical role has probably hindered exploration of BST-2 antiviral function in vivo. We previously reported that BST-2 tethers Chikungunya virus-like particle on the cell plasma membrane. To explore the role of BST-2 in Chikungunya virus (CHIKV) replication and host protection, we utilized Chikungunya virus strain 181/25 to examine early events during CHIKV infection in BST-2-/- mouse model. We observed interesting dichotomy between wild-type (WT) and BST-2-/- mice. BST-2 deficiency increased inoculation site viral load, culminating in higher systemic viremia and increased lymphoid tissues tropism. Suppressed inflammatory innate response demonstrated by impaired IFNaalpha;, IFN, and CD40 ligand (CD40L) expression was observed in BST-2-/- mice compared to the WT controls. These findings suggest that, in part, BST-2 protects lymphoid tissues from CHIKV infection and regulates CHIKV-induced inflammatory response by the host.
Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (Infectious-Subgenomic-Amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.
The human herpesviruses (HHVs) are remarkably successful human pathogens, with some members of the family successfully establishing infection in the vast majority of humans in the world. Although many HHV infections result in asymptomatic infection or mild disease, there are rare cases of severe disease and death found with nearly every HHV. Many of the pathogenic mechanisms of these viruses are poorly understood, and in many cases effective anti-viral drugs are lacking. Only a single vaccine exists for the HHVs, and researchers have been unable to develop treatments to cure the persistent infections associated with HHVs. A major hindrance to HHV research has been the lack of suitable animal models, with the notable exception of the herpes simplex viruses. One promising area for HHV research is the use of humanized mouse models, in which human cells or tissues are transplanted into immunodeficient mice. Current humanized mouse models mostly transplant human hematopoietic stem cells (HSCs), resulting in the production of a variety of human immune cells. Although all HHVs are thought to infect human immune cells, the beta- and gammaherpesviruses extensively infect and establish latency in these cells. Thus, mice humanized with HSCs hold great promise to study these herpesviruses. In this review, we provide historical perspective on the use of both older and newer humanized mouse models to study HHV infections. The focus is on current developments in using humanized mice to study mechanisms of HHV-induced pathogenesis, human immune responses to HHVs, and effectiveness of anti-viral drugs.
Whole virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2) and its reassortant strains H3N4, H3N5, H3N7, which have the same hemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent concentrations of each vaccine (1 to 0.01 mmicro;g/mouse). All of the mice that received the highest concentration of each vaccine (1 mmicro;g/mouse) showed equivalent high hemagglutinin inhibition (HI) antibody titers of over 1:40, and survived the H3N2 challenge viruses. However, mice that received lower concentrations of vaccine (0.1 or 0.01 mmicro;g/mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributes to the protection, especially when the HI antibody level is low. These results suggest that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.
Direct acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown up-regulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell culture model. Here we investigated whether CB1 blockade inhibits HCV replication. The antiviral effect of a CB1 antagonist, AM251 was examined in the JFH1 cell culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 shRNA was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70%), viral protein (~80%), the production of new virus particles (~70%), and virus infectivity (~90%). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARaalpha;). This effect was mediated by AMPK. Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA, compared with controls. Reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drugs with activity against HCV.
In 2012, a mutant porcine circovirus type 2 (mPCV2) strain was identified in cases of porcine circovirus associated disease (PCVAD) in the United States. The mPCV2 has an additional amino acid, Lysine (K), in the capsid at position 234. The objectives of this study were to compare the pathogenicity of mPCV2, PCV2a and PCV2b in pigs using biologically pure infectious virus stocks derived from respective infectious DNA clones and to investigate the importance of genotype-specific open reading frames (ORF) 2 and presence of K at position 234 of the capsid. A total of 47, two-week-old, caesarian-derived, colostrum-deprived (CDCD) pigs were assigned to one of 7 groups. At 3 weeks of age, the pigs were experimentally inoculated with saline, PCV2a, PCV2b, mPCV2, PCV2b-234-K (Lysine addition in ORF2), chimeric PCV2b-ORF1/mPCV2-ORF2, or reciprocal chimeric mPCV2-ORF1/PCV2b-ORF2. All pigs were necropsied 21 days post infection (dpi). Gross lesions were limited to visible icterus and loss of body condition in a portion of the mPCV2 pigs. The amount of PCV2 DNA was significantly higher in pigs inoculated with mPCV2 compared to PCV2b in sera at 7 dpi and fecal swabs at 14 dpi. Based on lymphoid lesions, a higher prevalence of PCVAD was seen in pigs infected with PCV2's containing the additional 234-K (64.3%) compared to those infected with a PCV2 with the regular 233-bp ORF2 (40%). Results indicate that all PCV2 isolates were capable of inducing severe lesions and disease in the CDCD pig model, and there was no significant difference in virulence.
Astroviruses infect human and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive bred porcupines and nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of complete ORF2 sequence suggested that it may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astrovirus in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.
The objective of this study was to compare the virulence and pathogenicity of a combination of concurrent infections of two genotypes of porcine circovirus type 2 (PCV2) and two genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) in terms of PCV2 viremia, and PCV2-associated lesions and antigens in co-infected pigs. Co-infection induced significantly lower levels of anti-PCV2 and anti-PRRSV IgG antibodies than infection with one genotype alone, regardless of the genotype of the two viruses. Pigs with PCV2a (or 2b)/type 1 PRRSV had significantly (P llt; 0.05) higher levels of PCV2 viremia, more severe PCV2-associated lesions, and more PCV2 DNA within the lesions compared to pigs with PCV2a (or 2b)/type 1 PRRSV. However, there was no significant difference in these parameters in pigs with PCV2a/type 2 PRRSV or PCV2b/type 2 PRRSV. The results of this study demonstrate significant differences in the virulence and pathogenicity of type 1 and type 2 PRRSV but no significant differences in the virulence and pathogenicity of PCV2a and PCV2b with respect to the production of PCV2-associated lesions.
Begomoviruses are whitefly-transmitted, single-stranded DNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as 'mixing vessels' where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared to cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, P. lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were intensively sampled from two regions across Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. The MaYSV population (N = 99) was more variable than the BGMV population (N = 147), which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography.
A high prevalence of the rtI187V polymerase substitution of hepatitis B virus (HBV) was detected in nucleos(t)ide analogs-naiiuml;ve and -treated chronic hepatitis B (CHB) patients. We aimed at assessing the replicative capacity and susceptibility of rtI187V alone or in conjunction with lamivudine (LAM) or adefovir (ADV) -resistant mutations to LAM and ADV in vitro. The reverse transcriptase region of HBV isolates was directly sequenced from a cohort of 300 CHB patients from China. Replication-competent HBV constructs containing rtI187V and combined with LAM-resistant (rtM204I, rtL180M/rtM204V) mutations were generated, and compared with wild-type (WT), LAM-resistant single (rtM204I) or double (rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. In a Chinese cohort of 300 CHB patients, 8.7% (26/300) showed substitution in the rtI187 with V. Of note, the rtI187V prevalence in genotype B was significant higher than in genotype C (95.2 vs. 4.8%). In vitro phenotypic assays showed that the viruses bearing the rtI187V had impaired replication efficacy when compared to WT and the virus carrying the rtI187V combined with LAM-resistant single or double mutations showed even more significantly impaired replicative capacities. Furthermore, rtI187V remained susceptible towards treatment with LAM or ADV in vitro whereas the combination of rtI187V substitution with LAM-resistant mutations was resistant to LAM but still sensitive to ADV. Our study reveals that the rtI187V substitution in the HBV polymerase frequently occurred in CHB patients particular with genotype B. However, the emergence of rtI187V substitution significantly impair viral replication but without affecting drug sensitivity in vitro.
Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1 based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated sub-genomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted chimaeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full length and bipartite systems may provide further insight into this process.
Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPSc in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Nonhuman primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPSc by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPC substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPSc in the CSF by serial PMCA, and the CSF levels of PrPSc tended to increase with disease progression. In addition, PrPSc was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPSc in nonhuman primate models of CJD.
Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to man. Severe hantavirus infection is manifested by impairment of renal function, pulmonary edema, and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis but the underlying mechanisms are not fully understood. Here we describe that galectin-3 binding protein (Gal-3BP) is upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments show that while Vero E6 cells (African green monkey kidney cell line) constitutively express and secrete Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques experimentally infected with hantavirus indicated that hantavirus infection induces Gal-3BP also in vivo. Finally, the analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than convalescent phase. Furthermore, the Gal-3BP levels in HFRS patients correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.
JS11C1, a member of putative new subgroup of avian leukosis virus different from all six known subgroups from chickens based on Gp85 amino acid sequence comparison, was isolated from Chinese native chicken breeds in 2012. In order to further study the genome structure, biological characteristics, and the evolutionary relationship of the virus with others of known subgroups infected chickens, we determined the complete genome sequence, constructed an infectious clone of ALV strain JS11C1, and performed comparison analysis using the whole genome sequence or elements with that of other ALSVs all available in GenBank. The results showed that the full-length sequence of the JS11C1 DNA provirus genome was 7707 bp, which is consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The rescued infectious clone of JS11C1 showed similar growth rate and biological characteristics to its original virus. All the comparison analysis based on whole genomes support the opinion that the new isolates are relatively far related to any known subgroups of ALVs and might be classified as a new subgroup.
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for the systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with an enhanced green fluorescent protein (EGFP) fused to the nucleocapsid to monitor the entry of virus into primary midgut epithelium cells ex vivo by confocal fluorescence microscopy. The EGFP-labeled virus showed the same BV virulence and ODV infectivity as wild type virus. The ability to bind and enter host cells was then visualized for wild type AcMNPVs and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, while PIF1 and PIF2 play important roles in entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and northeastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time that primary human astrocytes are sensitive to TBEV infection, although the infection does not affect their viability. The infection induces a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key proinflammatory cytokines/chemokines (e.g., tumor necrosis factor aalpha;, interferon aalpha;, interleukin (IL)-1bbeta;, IL-6, IL-8, interferon -induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) is upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 nm (pplusmn; 0.15 nm) diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of proinflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood-brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.
Laboratory animal models have provided valuable insight into foot-and-mouth disease virus (FMDV) pathogenesis in epidemiologically important target species. While not perfect, these models have delivered an accelerated time frame to characterize the immune responses in natural hosts and a platform to evaluate therapeutics and vaccine candidates at a reduced cost. Further expansion of these models in mice has allowed access to genetic mutations not available for target species, providing a powerful and versatile experimental system to interrogate the immune response to FMDV and to target more expensive studies in natural hosts. The purpose of this review is to describe commonly used FMDV infection models in laboratory animals and to cite examples when these models have failed or successfully provided insight relevant for target species, with an emphasis on natural and vaccine induced immunity.
The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein, close to the VP1/2A cleavage site, inhibited cleavage of this junction and produced "self-tagged" virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus (Gullberg et al. (2013) J Virol 87, 11591nndash;11603). It is now shown that introduction of this E83K change alone, into a serotype O virus, resulted in the rapid accumulation of a second site substitution within the 2A sequence (L2P) which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained the uncleaved VP1-2A protein; the 2A L2P substitution resulted in the VP1/2A junction being totally resistant to cleavage by the 3C protease and hence may be a preferred route for "tagging" virus particles.
Alternative methods to the standard hemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus hemagglutinin (HA) were developed in this study. Vaccinia virus recombinants expressing reference HAs were used to immunize rabbits from which polyclonal antibodies were obtained. These antibodies were subtype specific but showed limited intra-subtype strain specificity in ELISA. The discriminatory capacity of these antibodies was however markedly increased after adsorption to cells infected with heterologous influenza viruses, revealing antigenic differences that were otherwise undistinguishable by standard HI and neutralization tests. Furthermore, the unadsorbed antibodies could be used to select escape mutants of the reference strain which after sequencing unveiled amino acid changes responsible of the noted antigenic differences. These procedures therefore provide alternative methods for the antigenic characterization of influenza HA and might be useful in studies of HA antigenic evolution.
The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.
Influenza B viruses have become increasingly more prominent during influenza seasons. Influenza B species is typically considered a mild disease that receives less attention than influenza A species, but has been causing 20% to 50% of the total influenza incidence in several regions around the world. Although there is increasing evidence of mid to lower respiratory tract diseases such as bronchitis and pneumonia in influenza B patients, little is known about the pathogenesis of recent influenza B viruses. Here we investigated the clinical and pathological profiles of infection with strains representing the two current co-circulating B lineages (B/Yamagata and B/Victoria) in the ferret model. Specifically, we studied two B/Victoria (B/Brisbane/60/2008 and B/Bolivia/1526/2010) and two B/Yamagata (B/Florida/04/2006 and B/Wisconsin/01/2010) strain infections in ferrets and observed strain-specific but not lineage-specific pathogenicity. We found B/Brisbane/60/2008 caused the most severe clinical illness and B/Brisbane/60/2008 and the B/Yamagata strains instigated pathology in the middle to lower respiratory tract. Importantly, B/Brisbane/60/2008 established efficient lower respiratory tract infection with high viral burden. Phylogenetic analyses demonstrated profound reassortment among recent influenza B viruses which indicated the genetic make-up of B/Brisbane/60/2008 differed from the other strains and resembled the 2002-2003 influenza B global outbreak strain, B/Brisbane/32/2002. This may explain the pathogenicity difference post infection in ferrets. Our study characterized influenza B infections in ferrets and this model may aid in the future development of effective influenza therapeutic treatment.
The cell line IPLB-LD-652Y derived from the gypsy moth (Lymantria dispar, Linn.) is routinely used to study insect virus-host interactions. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered persistently infecting this cell line. LdIV1 belongs to the genus Iflavirus. LdIV1 has icosahedral particles of approximately 30 nm in diameter and a 10,044 nucleotide polyadenylated, positive sense RNA genome encoding a predicted polyprotein of 2,980 amino acids. LdIV1 was induced by a viral suppressor of RNA silencing suggesting that acute infection is restricted by RNAi. We detected LdIV1 in all tested tissues of gypsy moth larvae and adults but the virus was absent from other L. dispar nndash; derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group; many of which covertly infect their hosts.
Human Cytomegalovirus (HCMV) infection and reactivation is a major cause of morbidity in immuno-suppressed transplant patients and late stage AIDS sufferers. Interestingly, epidemiological studies have shown that the immuno-suppressive regimen used can have differential prognoses for patients regarding HCMV infection and disease. Specifically, patients administered Sirolimus (rapamycin) exhibit more favourable outcomes, suggesting that rapamycin has activity against HCMV in vivo. Given the relative lack of activity against lytic infection it has been postulated rapamycin may inhibit HCMV reactivation. Here we show that rapamycin given acutely or chronically has little impact on the induction of HCMV reactivation in experimentally latent dendritic cells or cells from naturally latent individuals. Furthermore, we extend these observations to include other inhibitors of mTORC1 and mTORC 2 which similarly have minimal effect on the induction of IE gene expression from latency. Taken together, these data suggest that favourable outcomes associated with sirolimus may be due to indirect effects that influence HCMV reactivation rather than a direct mechanistic action against HCMV itself.
The Orbivirus genus of the family Reoviridae is comprised of 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographic areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analyses by electron microscopy and gel electrophoresis, respectively revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analyses support previous serologic findings, which indicated that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, 6 out of 13 analyzed CGLV serotypes show reassortment of their genome segments.
The mechanism used by Bluetongue Virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we have investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was sequentially mutated; and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA suggested that a conformational motif formed by interaction of the 5rrsquo; and the 3rrsquo;ends of the molecule is necessary and sufficient for packaging. A similar structural signal was also identified in segment 8 (S8) of serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was successfully packaged, confirming that the motif identified directs the correct packaging of the segment.
Porcine hemagglutinating encephalomyelitis virus (PHEV) is the main causative agent of porcine coronavirus-associated disease, which is characterized by encephalomyelitis and involves the central nervous system. However, little is known about the molecular mechanisms of brain injury. To gain insight into the interaction between the virus and host cells, global gene expression changes in the cerebral cortex of PHEV- or mock-infected mice were investigated using DNA microarray analysis and quantitative real-time PCR. The results of the microarray analysis showed that 365 genes on day 3 post infection and 781 genes on day 5 post infection are differentially expressed in response to PHEV infection in the cerebral cortex. The up-regulated genes are mainly involved in immune system processes, antigen processing and presentation, the Jak-STAT signaling pathway, the RIG-I-like receptor signaling pathway, Toll-like receptor signaling and apoptosis-related proteases, etc. However, significantly down-regulated genes are mainly involved in nervous system development, synaptic transmission, neuron projection development, the transmission of nerve impulse, the negative regulation of glial cell differentiation, etc. The differential expression of these genes suggests a strong host antiviral response, but may also contribute to the pathogenesis of PHEV resulting in encephalomyelitis.
Bungowannah virus is the most divergent pestivirus, and both origin and reservoir host have not been identified so far. We therefore performed in vitro tropism studies, which showed that Bungowannah virus differs remarkably from other pestiviruses. Interestingly, cell lines of vervet monkey, mouse, human and even of bat origin were susceptible. This broad in vitro-tropism was not observed for a chimeric bovine viral diarrhea virus expressing all structural proteins of Bungowannah virus. The viral envelope was not sufficient to completely transfer the cell tropism of Bungowannah virus to another pestivirus, and viral RNA replication was markedly reduced or even not detectable in a number of different cell lines for the tested BVDV strain and the chimera. We therefore suggest that the replication machinery together with the viral envelope is responsible for the unique broad cell tropism of Bungowannah virus.
Background: Chronic hepatitis C (HCV) results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Methods: Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for M30 cytoDEATH and cleaved PARP. In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, and primary mouse (PMoH) and human hepatocytes (PHH). Cell viability and apoptosis were studied using crystal violet assays and Western immunoblots probed for cleaved caspase 3 and cleaved PARP, with and without treatment with Q-VD-Oph and necrostatin-1. Results: Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared to HCV-negative patients. rAdHCV infection reduced cell viability compared to uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoH and PHH infected with rAdHCV showed significantly increased levels of apoptotic markers compared to uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-Oph and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoH. Conclusions: Hepatocyte apoptosis is increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes and this was inhibited by Q-VD-Oph and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis and provide new insights into the mechanisms of HCV-induced liver injury.
This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks, and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation (p.i.). The titers of domestic duck viruses in chickens were much higher than those of wild bird viruses. Both domestic duck and wild bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titers in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting, and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.
Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 % and 11.5%, respectively, of those transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, an immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.
The flavivirus West Nile virus (WNV) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of flavivirus cDNA genomes with large internal deletions within capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus promoter (CMV) and C gene was placed under the control of a second copy of CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation we aimed at increasing SRIP production by optimizing trans-C expression via incorporating different forms of C and the use of a more powerful promoter. The EF1aalpha; promoter encoding an extended form of C was demonstrated to produce the highest titers of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titers were further improved via incorporation of a glycosylation motif in E protein. The optimized DNA yields ~100-fold greater titers of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.
Dengue virus belongs to Flavivirus and contains a positive-stranded RNA genome Binding of dengue virus to host cells was mediated through domain III of the viral envelope protein. Many therapeutic monoclonal antibodies (mAbs) against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 amino acids (V365NIEAEPPFG374) from domain III of the envelope protein in dengue virus serotype 2 (DENV2) was selected to design the primers for LAE technique. A DNA fragment encoding 10 directed repeats of these 10 amino acids for producing the tandem repeated peptides was obtained and fused it with GST-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from E. coli and used as antigen for immunizing rabbits to obtain polyclonal antibody. Furthermore, this EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using immunoblot and immunofluorescence assays. Most importantly, this antibody was also capable of detecting DENV2 virions by ELISA assay and could block viral entry into BHK-21 cells as shown by immunofluorescence and qRT-PCR assays. Taken together, LAE technique could be applied for production of antibody against antigen with low antigenicity successfully and carries a high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic application in the future.
Virions of the RPV strain of Cereal yellow dwarf virus (CYDV-RPV) were purified from infected oat tissue and analyzed by mass spectrometry. Two conserved residues, K147 and K181, residing in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favors acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.
Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in BFV are very similar to RRV. Although RRV disease has been extensively studied, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis and identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Herein to our knowledge we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to RRV. In comparison to RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-aalpha;, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understanding disease pathogenesis. With an increasing number of BFV cases occurring annually a better understanding of the disease mechanisms is essential for future therapeutic development.
Infection of macaques with live-attenuated SIV usually results in long-lasting efficient protection against infection with pathogenic immunodeficiency viruses. However, attenuation by deletion of regulatory genes such as nef is not complete, leading to a high viral load and fatal disease in some animals. To characterize immunological parameters and polymorphic host factors we studied 17 rhesus macaques infected with attenuated SIVmac239NU. Eight animals were able to control viral replication whereas the remaining animals (non-controllers) displayed variable set-point viral loads. Peak viral load at 2 weeks post infection correlated significantly with set-point viral load (pllt;0.0001). CD4+ T cell frequencies differed significantly soon after infection between controllers and non-controllers. Abnormal B-cell activation previously ascribed to Nef-function can be observed in non-controllers already 8 weeks after infection despite the absence of Nef. Two non-controllers developed an AIDS-like disease within 102 weeks post infection. Virus from these animals transmitted to naiiuml;ve animals replicated at low levels and the recipients did not develop immunodeficiency. This suggests that host factors determined differential viral load and subsequent disease course. Known MHC class I alleles associated with disease progression in SIV wild type infection only marginally influenced the viral load in nef-infected animals. Protection from SIVmac251 was associated with homozygosity for Mhc class II in conjunction with a TLR7 polymorphism and showed a trend with initial viral replication. We speculate that host factors whose effects are usually masked by Nef are responsible for the different disease courses in individual animals upon infection with nef-deleted viruses.
During infection the influenza A virus NS1 protein interacts with a diverse range of viral and cellular factors to antagonize host antiviral defences and promote viral replication. Here, I review the structural basis for some of these functions, and discuss the emerging view that NS1 cannot simply be regarded as a rrsquo;staticllsquo; protein with a single structure. Rather, the dynamic property of NS1 to adopt various quaternary conformations is critical for its multiple activities. Understanding NS1 plasticity, and the mechanisms governing this, will be essential for assessing both fundamental protein function and the consequences of strain-dependent polymorphisms in this important virulence factor.
Poxvirus vectors represent promising HIV vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant Capripoxvirus vector, Lumpy Skin Disease Virus (LSDV) in combination with Modified Vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrate that the combination regimen was immunogenic in rhesus macaques, inducing high magnitude, broad and balanced CD4+ and CD8+ T cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.
Several baculoviruses can induce host cell aggregation during infection; however, the molecular basis remains unknown. The Rho family of small GTPases, including Rho1, Racs and Cdc42, plays important roles in cell migration and cell-cell contact. Activated GTPases target actin polymerization to discrete sites on the plasma membrane, thereby inducing membrane protrusions. In this study, we demonstrated that Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection induced the amoeboid movement and aggregation of SpLi-221 cells in vitro. The amount of Rho1-GTP increased in the infected cells, which suggests that Rho1 was activated upon infection. RNA interference and superinfection of dominant-negative recombinants revealed that the SpltNPV-induced SpLi-221 cell aggregation was dependent on the Rho1, but not Racs or Cdc42, signaling pathway. Inhibition of Rho-associated protein kinase (Rok) activity by the inhibitor Y-27632 significantly reduced SpLi-221 cell aggregation. Silencing Rho1 expression with RNA interference decreased SpltNPV propagation by approximately 40% in vitro, when SpLi-221 cells were infected at a low, but not high, multiplicity of infection, suggesting that the SpltNPV-induced cell aggregation may benefit SpltNPV spread.
Key virus traits such as virulence and transmission strategies rely on genetic variation that result in functional changes in the interactions between hosts and viruses. Here comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host-virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates have similar genome size and content. Complete genome analyses of single nucleotide polymorphisms, insertions and deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus-host cells interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150), or secondary infections (ME35), and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbor high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways.
The full-length genome sequence of a porcine picobirnavirus detected in Italy in 2004 was determined. The S segment was 1730 nucleotide (nt) in length, coding for a putative RNA-dependent RNA polymerase. Two distinct sub-populations of L segment (LA and LB) were identified in the sample, with the sizes ranging from 2351 to 2666 nt. The ORF1 coding for a protein of unknown function, contained repetitions of the ExxRxNxxxE motif in variable number. The capsid protein coding ORF2 spanned nt 810-2447 in the LB variants and started at nt 734 in the LA variants. However, a termination codon was present only in one of all LA segment variants. Three dimensional modelling of the porcine PBV capsids suggested structural differences in the protruding domain, tentatively involved as antigens in humoral immune response. Altogether, these findings suggest simultaneous presence of two different picobirnavirus strains sharing the same S segment but displaying genetically diverse L segments. In addition, the sample probably contained a mixture of PBVs with aberrant RNA replication products. Altered structure in the L segments could be tolerated and retained in the presence of functionally integer cognate genes and represent a mechanism of virus diversification.