|Journal of General Virology current issue|
During infection, the influenza A virus non-structural protein 1 (NS1) 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 llsquo;staticrrsquo; 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 plasticity, will be essential for assessing both fundamental protein function and the consequences of strain-dependent polymorphisms in this important virulence factor.
The human herpesviruses (HHVs) are remarkably successful human pathogens, with some members of the family successfully establishing infection in the vast majority of humans worldwide. 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 antiviral 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 haematopoietic 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 a 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 antiviral drugs.
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 5nndash;10 days post-inoculation. The titres 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 titres 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.
Influenza B viruses have become increasingly more prominent during influenza seasons. Influenza B infection is typically considered a mild disease and receives less attention than influenza A, 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. Our phylogenetic analyses demonstrate profound reassortment among recent influenza B viruses, which indicates the genetic make-up of B/Brisbane/60/2008 differs from the other strains. This may explain the pathogenicity difference post-infection in ferrets.
Alternative methods to the standard haemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus haemagglutinin (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.
Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, 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 to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of 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 that of RRV. In comparison with 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 understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development.
Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the 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 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.
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 that released from cells 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, 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.
West Nile virus (WNV; genus Flavivirus, family Flaviviridae) 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 cDNA copies of flavivirus RNA genomes with large internal deletions within the 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 (CMV) promoter and the C gene was placed under the control of a second copy of the 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 construct containing an elongation factor EF1aalpha; promoter encoding an extended form of C was demonstrated to produce the highest titres of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titres were further improved via incorporation of a glycosylation motif in the envelope protein. The optimized DNA yielded ~100-fold greater titres of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.
Porcine haemagglutinating encephalomyelitis virus (PHEV) is the main causative agent of porcine coronavirus-associated disease, which is characterized by encephalomyelitis and involves the central nervous system. Little is known about the molecular mechanisms of brain injury caused by PHEV. To gain insight into the interaction between the virus and host cells, changes in global gene expression 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 (p.i.) and 781 genes on day 5 p.i. were differentially expressed in response to PHEV infection in the cerebral cortex. The upregulated genes were mainly involved in immune system processes, antigen processing and presentation, the Jaknndash;STAT signalling pathway, the RIG-I-like receptor signalling pathway, Toll-like receptor signalling and apoptosis-related proteases. Significantly downregulated genes were mainly involved in nervous-system development, synaptic transmission, neuron-projection development, the transmission of nerve impulses and negative regulation of glial cell differentiation. The differential expression of these genes suggests a strong antiviral host response, but may also contribute to the pathogenesis of PHEV resulting in encephalomyelitis.
Chronic hepatitis C virus (HCV) infection 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. Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for cytokeratin 18 (M30 CytoDEATH) and cleaved poly(ADP-ribose) polymerase (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, primary mouse hepatocytes (PMoHs) and primary human hepatocytes (PHHs). Cell viability and apoptosis were studied using crystal violet assays and Western blots probed for cleaved caspase-3 and cleaved PARP, with and without treatment with the pan-caspase inhibitor Q-VD-OPh and necrostatin-1. Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared with HCV-negative patients. rAdHCV infection reduced cell viability compared with uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoHs and PHHs infected with rAdHCV showed significantly increased levels of apoptotic markers compared with 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 PMoHs. Hepatocyte apoptosis is known to be 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.
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 diarrhoea virus (BVDV) 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 either markedly reduced or 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.
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.
The full-length genome sequence of a porcine picobirnavirus (PBV) detected in Italy in 2004 was determined. The smaller (S) genome segment was 1730 nt, coding for a putative RNA-dependent RNA polymerase. Two distinct subpopulations of larger (L) genome 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 a variable number of repetitions of the ExxRxNxxxE motif. The capsid protein-coding ORF2 spanned nt 810nndash;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 the LA segment variants. Three-dimensional modelling of the porcine PBV capsids suggested structural differences in the protruding domain, tentatively involved as antigens in the humoral immune response. Altogether, these findings suggest the simultaneous presence of two different PBV 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 represents a mechanism of virus diversification.
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 investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially 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 followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5' and 3' ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV 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 packaged successfully, confirming that the motif identified directs the correct packaging of the segment.
The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical 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 analysis 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 analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.
Human cytomegalovirus (HCMV) infection and reactivation are a major cause of morbidity in immune-suppressed patients. Interestingly, epidemiological studies have shown that patients administered the mammalian target of rapamycin (mTOR) inhibitor, sirolimus (rapamycin), exhibit more favourable outcomes, suggestive of activity against HCMV in vivo. Given its relative lack of activity against lytic infection, it is postulated that rapamycin inhibits HCMV reactivation. Here, we showed that rapamycin administered acutely or chronically has little impact on induction of immediate early (IE) gene expression in experimentally latent dendritic cells or cells from naturally latent individuals. Furthermore, we extended these observations to include other inhibitors of mTORC1 and mTORC 2, which similarly have minimal effects on induction of IE gene expression from latency. Taken together, these data suggest that favourable outcomes associated with sirolimus are attributable to indirect effects that influence HCMV reactivation, rather than a direct mechanistic action against HCMV itself.
Poxvirus vectors represent promising human immunodeficiency virus (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 demonstrated 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.
Infection of macaques with live attenuated simian immunodeficiency virus (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 (p.i.) 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 could already be observed in non-controllers 8 weeks after infection despite the absence of Nef. Two non-controllers developed an AIDS-like disease within 102 weeks p.i. Virus from these animals transmitted to naiiuml;ve animals replicated at low levels and the recipients did not develop immunodeficiency. This suggested that host factors determined differential viral load and subsequent disease course. Known Mhc class I alleles associated with disease progression in SIV WT 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 speculated that host factors whose effects were usually masked by Nef were responsible for the different disease courses in individual animals upon infection with nef-deleted viruses.
The cell line IPLB-LD-652Y, derived from the gypsy moth (Lymantria dispar L.), is routinely used to study interactions between viruses and insect hosts. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered to persistently infect IPLB-LD-652Y. LdIV1 belongs to the genus Iflavirus. LdIV1 formed icosahedral particles of approx. 30 nm in diameter and contained a 10 044 nt polyadenylated, positive-sense RNA genome encoding a predicted polyprotein of 2980 aa. LdIV1 was induced by a viral suppressor of RNA silencing, suggesting that acute infection is restricted by RNA interference (RNAi). We detected LdIV1 in all tested tissues of gypsy-moth larvae and adults, but the virus was absent from other L. dispar-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 of viruses, many of which covertly infect their hosts.
Key virus traits such as virulence and transmission strategies rely on genetic variation that results 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 hostnndash;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 had a similar genome size and content. Complete genome analyses of single nucleotide polymorphisms and insertions/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. Virusnndash;host cell 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 harbour 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 that the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways.
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 cellnndash;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 suggested 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, signalling 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, m.o.i., suggesting that the SpltNPV-induced cell aggregation may benefit SpltNPV spread.
Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, 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 favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.
|Journal of General Virology Publish Ahead of Print|
Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment 4 (S4) showed that it consists of 3410 nucleotides with a single ORF of 1110 amino acids which could encode a protein of ~ 127 kDa (p127). Bioinformatics analysis showed the presence of 5rrsquo; RNA triphosphatase (RTPase) domain (LRDR), S-adenosyl-L-methionine (SAM) binding (GxGxG) motif and K-D-K-E tetrad of 2rrsquo;-O-methyltransferase (MTase), which suggests that S4 may encode RTPase and MTase. The ORF of S4 was expressed in E. coli as His-tagged fusion protein and purified by Ni-NTA affinity chromatography. Biochemical analysis of recombinant p127 showed its 5rrsquo; RTPase as well as SAM dependent guanine N-7 and ribose 2rrsquo;-O-MTase activity. MTase assay using in vitro transcribed AmCPV S2 RNA having 5rrsquo; G*pppG end showed that guanine N-7- methylation occurs prior to the ribose 2rrsquo;-O-methylation to yield m7GpppG/ m7GpppGm RNA cap.. Mutagenesis of the SAM binding (GxGxG) motif (G831A) completely abolished N-7 and 2rrsquo;-O-MTase activity indicating importance of these residues for capping. From the kinetic analysis the Km of N-7-MTase for SAM and RNA was calculated as 4.41 and 0.39 mmu;M, respectively. These results suggest that AmCPV S4 encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5rrsquo; end of viral RNA.
Bats have been found to harbor a number of new emerging viruses with zoonotic potential and there has been a great deal of interest in identifying novel bat pathogens to determine risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid, however virus isolation is still a challenge and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports we have described the isolation of Hendra virus, Menangle virus and Cedar virus, in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.
The interferon (IFN) immune system plays an essential role in protecting the host against most viral infections. In order to explore the interactions between the IFN pathway and Respiratory syncytial virus (RSV) infection, and to identify potential IFN-stimulated genes (ISGs) that may be involved in suppressing the replication of RSV, we utilized an IFN pathway-specific microarray to study the effects of RSV infection on the IFN pathway in HeLa cells. We showed that RSV infection enhanced the expression of a series of ISGs, including oligoadenylate synthetase 2 (OAS2), interferon-induced transmembrane protein 1 (IFITM1) and myxovirus-resistance 2 (Mx2). Our results also showed that the IFITM proteins potently inhibited RSV infection mainly by interfering with both virus entry and the subsequent replication steps, but not the attachment process. The anti-viral effect of IFITM3 protein was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expressions of IFITM1 and IFITM3 proteins facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of interferon regulatory factor-3 (IRF3) through interfering with the detection of viral RNA by the melanoma differentiation-associated gene 5 (MDA5) and the retinoic acid-inducible gene I (RIG-I). These results demonstrated that the restriction of RSV infection by the IFITM proteins was achieved through the inhibition of virus entry and replication, and they provide further insight for exploring the mechanism of IFITM proteins-mediated virus restriction.
Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumor virus (MMTV) is gaining acceptance as possibly being associated with human breast cancer and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkage to APOBEC3 suggested.
Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we showed that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis, and immune responses in the liver of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver, and serum AST, ALT, ALP, GGT, and T-Bil activities were greatly up-regulated, and ducklings died at 36 hours post-infection (h.p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen, and intestine, while the SH strain replicated more efficiently than FC64. IFN-, IL-2, iNOS, and NO were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-aalpha;, IFN-bbeta;, IFITM1, ISG12, OASL, and IL-6 were moderately induced by SH infection at 24 h.p.i., and dramatically induced by FC64 infection at 36 h.p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of anti-viral antibodies within 45 days post-infection. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study.
Human herpesvirus 6 (HHV-6) glycoprotein M (gM) is an envelope glycoprotein that associates with glycoprotein N (gN), forming the gM/gN protein complex, as similar manner to the other herpesviruses. LC-MS/MS analysis showed that the HHV-6 gM/gN complex interacts with the v-SNARE protein, VAMP3. VAMP3 colocalized with the gM/gN complex at the trans-Golgi network and the other compartments, possibly late endosome in HHV-6-infected cells, and its expression gradually increased during the late phase of virus infection. Finally, VAMP3 was incorporated into mature virions, maybe being transported with the gM/gN complex.
The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the remaining N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified. The domain was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain was not reacted with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.
Replicon particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR) and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) are able to produce high levels of NSR particles. In addition, using reverse-genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains 2 amino acid substitutions in respectively Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.
Despite the success of combined antiretroviral therapy in controlling viral replication in HIV-infected individuals, HIV-associated neurocognitive disorders (Manganaro et al., 2014), commonly referred to as neuroAIDS, remain a frequent and poorly understood complication. Infection of CD8+ lymphocyte-depleted rhesus macaques with SIVmac251 viral swarm is a well-established rapid disease model of neuroAIDS that has provided critical insight into HAND onset and progression. However, no studies so far have characterized in depth the relationship between intra-host viral evolution and pathogenesis in this model. SIV env gp120 sequences were obtained from six infected animals. Sequences were sampled longitudinally from several lymphoid and non-lymphoid tissues, including individual lobes within the brain at necropsy, for four macaques; two animals were sacrificed at 21 days post-infection (dpi) to evaluate early viral seeding of the brain. Bayesian phylodynamic and phylogeographic analyses of the sequence data were used to ascertain viral population dynamics and gene flow between peripheral and brain tissues, respectively. A steady increase in viral effective population size, with a peak occurring at approximately 50-80 dpi, was observed across all longitudinally monitored macaques. Phylogeographic analysis indicated continual viral seeding of the brain from several peripheral tissues throughout infection, with the last migration event before terminal illness occurring in all macaques from cells within the bone marrow. The results strongly support the role of infected bone marrow cells in HIV/SIV neuropathogenesis. In addition, our work demonstrates the applicability of Bayesian phylogeography to intra-host studies in order to assess the interplay between viral evolution and pathogenesis.
A new nodavirus (named covert mortality nodavirus, CMNV) is associated with covert mortality disease of shrimp which caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by RT-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in muscle and lymphoid organ. The tubular epithelium of hepatopancreas show significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shares 54%, 53% and 39% identities with the amino acid sequences of RNA-dependent RNA polymerase from flock house virus, black beetle virus, and MrNV. The results of fluorescence in situ hybridization (FISH) showed that the hepatopancreas, striated muscle, and lymphoid organ were positively reacting tissues. The average size of negative stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV positive.
Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, while Bovine Papillomavirus-1 (BPV-1) primarily causes warts in its natural host the cow, it can also cause locally aggressive and invasive skin tumours in equids known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here we describe the first phylogenetic analysis of BPV-1 in equine sarcoids, to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implies that the genetic diversity in the LCR variants is ancient and predates domestication of both equids and cattle. The phylogeny demonstrates geographic segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. While our data are consistent with BPV-1 originating in cattle, we find evidence of multiple, likely relatively recent, cross-species transmission events into horses. We also demonstrate the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate this variant shows a fitness advantage in equids. Our results add to the accumulating evidence that host specificity in papillomaviruses is more flexible than previously assumed.
Most viruses express one or several proteins that counter the anti-viral defences of the host-cell. This is the task of non-structural protein NS1 in influenza viruses. Absent from the viral particle but highly expressed in the infected cell, NS1 dramatically inhibits the cellular gene expression and prevents the activation of key players in the interferon system. In addition, NS1 selectively enhances the translation of viral mRNAs and may regulate the synthesis of viral RNAs. Our knowledge of the virus and of its protein NS1 has dramatically increased during the last fifteen years. The atomic structure of NS1 has been determined, many cellular partners were identified and its multiple activities have been studied in depth. This review presents our current knowledge and attempts to establish relationships between the RNA sequence, the structure of the protein, its ligands, its activities and the pathogenicity of the virus. A better knowledge of NS1 could help in elaborating novel antiviral strategies, based on either live vaccines with altered NS1, or on small-compounds inhibitors of NS1.
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.
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.