- Volume 92, Issue 12, 2011
Volume 92, Issue 12, 2011
- Review
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How do plant viruses induce disease? Interactions and interference with host components
More LessPlant viruses are biotrophic pathogens that need living tissue for their multiplication and thus, in the infection–defence equilibrium, they do not normally cause plant death. In some instances virus infection may have no apparent pathological effect or may even provide a selective advantage to the host, but in many cases it causes the symptomatic phenotypes of disease. These pathological phenotypes are the result of interference and/or competition for a substantial amount of host resources, which can disrupt host physiology to cause disease. This interference/competition affects a number of genes, which seems to be greater the more severe the symptoms that they cause. Induced or repressed genes belong to a broad range of cellular processes, such as hormonal regulation, cell cycle control and endogenous transport of macromolecules, among others. In addition, recent evidence indicates the existence of interplay between plant development and antiviral defence processes, and that interference among the common points of their signalling pathways can trigger pathological manifestations. This review provides an update on the latest advances in understanding how viruses affect substantial cellular processes, and how plant antiviral defences contribute to pathological phenotypes.
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- Plant
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The N-terminal 12 amino acids of tomato aspermy virus 2b protein function in infection and recombination
More LessThe roles for various regions of the 2b protein in infection, hypervirulence and recombination were examined by introducing stop codons in a chimeric virus containing RNA 1 from the cucumber mosaic virus (CMV strain Q), RNA 3 from the tomato aspermy virus (TAV) and RNA 2 of CMV with a 2b gene from TAV. Chimeric virus expressing the intact 2b protein induced severe symptoms in inoculated Nicotiana clevelandii and Nicotiana glutinosa and facilitated CMV–TAV recombination, while chimeric viruses not expressing 2b protein did not infect plants systemically. Chimeric viruses expressing either the N-terminal 43 or 12 aa of the 2b protein infected both plant species systemically and facilitated CMV–TAV recombination, but induced mild symptoms and no symptoms in the infected plants, respectively. These data suggest that oligopeptides can have important functions in the biology of viruses and prompt a re-examination of existing small ORFs in sequenced virus genomes.
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Detection of plum pox potyviral protein–protein interactions in planta using an optimized mRFP-based bimolecular fluorescence complementation system
More LessIn previous studies, protein interaction maps of different potyviruses have been generated using yeast two-hybrid (YTH) systems, and these maps have demonstrated a high diversity of interactions of potyviral proteins. Using an optimized bimolecular fluorescence complementation (BiFC) system, a complete interaction matrix for proteins of a potyvirus was developed for the first time under in planta conditions with ten proteins from plum pox virus (PPV). In total, 52 of 100 possible interactions were detected, including the self-interactions of CI, 6K2, VPg, NIa-Pro, NIb and CP, which is more interactions than have ever been detected for any other potyvirus in a YTH approach. Moreover, the BiFC system was shown to be able to localize the protein interactions, which was typified for the protein self-interactions indicated above. Additionally, experiments were carried out with the P3N-PIPO protein, revealing an interaction with CI but not with CP and supporting the involvement of P3N-PIPO in the cell-to-cell movement of potyviruses. No self-interaction of the PPV helper component–proteinase (HC-Pro) was detected using BiFC in planta. Therefore, additional experiments with turnip mosaic virus (TuMV) HC-Pro, PPV_HC-Pro and their mutants were conducted. The self-interaction of TuMV_HCpro, as recently demonstrated, and the self-interaction of the TuMV_ and PPV_HC-Pro mutants were shown by BiFC in planta, indicating that HC-Pro self-interactions may be species-specific. BiFC is a very useful and reliable method for the detection and localization of protein interactions in planta, thus enabling investigations under more natural conditions than studies in yeast cells.
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- Animal
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- DNA viruses
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Transcriptional profiling of host gene expression in chicken liver tissues infected with oncogenic Marek’s disease virus
Marek’s disease virus (MDV), one of the most potent oncogenic herpesviruses, leads to highly contagious immunosuppressive and neoplastic disease in susceptible chickens. Previous studies mainly focused on the roles of host genes modulated by MDV in the virological rather than the neoplastic stage of disease. To investigate the molecular mechanisms of tumorigenesis in Marek’s disease further, a microarray analysis with Affymetrix Gene-Chip Chicken Genome Arrays was performed in a non-lymphoid tissue liver during the neoplastic stage. Of the 32 773 chicken transcriptions arrayed on a chip, 269 genes were significantly differentially expressed during the neoplastic stage caused by MDV infection (upregulated, 175; downregulated, 94). The altered genomic expression of 15 randomly selected genes was confirmed by real-time RT-PCR. Biological functions and pathways of the group of 269 differentially expressed genes were analysed by using a bioinformatics tool (ipa, Ingenuity Pathway Analysis). The results revealed that 19 possible gene networks with intermolecular connections and 22 significant metabolic and signalling pathways (P≤0.05) among 137 differentially expressed genes. These 137 genes were classified into a number of functional groups that included genetic disorder, cancer, cellular growth and proliferation, and cell death. In summary, the investigation of global host-gene expression, providing the biological functions of differentially expressed genes in lymphoid tumours of the liver in response to MDV infections, may contribute to a basic understanding of the molecular mechanisms involved in tumorigenesis following MDV infection.
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Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34
Release of herpes simplex virus type 1 (HSV-1) nucleocapsids from the host nucleus relies on the nuclear egress complex consisting of the two essential proteins pUL34 and pUL31. The cytoplasmically exposed N-terminal region of pUL34 interacts with pUL31, while a hydrophobic region followed by a short luminal part mediates membrane association. Based on its domain organization, pUL34 was postulated to be a tail-anchor (TA) protein. We performed a coupled in vitro transcription/translation assay to show that membrane insertion of pUL34 occurs post-translationally. Transient transfection and localization experiments in mammalian cells were combined with HSV-1 bacterial artificial chromosome mutagenesis to reveal the functional properties of the essential pUL34 TA. Our data show that a minimal tail length of 15 residues is sufficient for nuclear envelope targeting and pUL34 function. Permutations of the pUL34 TA with orthologous regions of human cytomegalovirus pUL50 or Epstein–Barr virus pBFRF1 as well as the heterologous HSV-1 TA proteins pUL56 or pUS9 or the cellular TA proteins Bcl-2 and Vamp2 revealed that nuclear egress tolerates TAs varying in sequence and hydrophobicity, while a non-α-helical membrane anchor failed to complement the pUL34 function. In conclusion, this study provides the first mechanistic insights into the particular role of the TA of pUL34 in membrane curving and capsid egress from the host nucleus.
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Infection with cytomegalovirus but not herpes simplex virus induces the accumulation of late-differentiated CD4+ and CD8+ T-cells in humans
Human cytomegalovirus (CMV) establishes persistent, usually asymptomatic, infection in healthy people. Because CMV infection is associated with the presence of lower proportions of peripheral naïve CD8+ T-cells and a higher fraction of late-differentiated CD8+ cells, commonly taken as biomarkers of age-associated compromised adaptive immunity (‘immunosenescence’), we asked whether chronic exposure to any persistent virus mediates these effects. Herpes simplex virus (HSV) is also a widespread herpesvirus that establishes lifelong persistence, but, unlike CMV, its impact on the distribution of T-cell subsets has not been established. Here, we analysed T-cell subsets in 93 healthy people aged 42–81 years infected or not infected with CMV and/or HSV. Individuals harbouring CMV were confirmed to possess lower frequencies of naïve CD8+ T-cells (defined as CD45RA+CCR7+CD27+CD28+) and greater proportions of late-differentiated effector memory (CD45RA−CCR7−CD27−CD28−) and so-called TEMRA (CD45RA+CCR7−CD27−CD28−) CD4 and CD8 subsets, independent of HSV seropositivity. In CMV-seronegative donors, HSV did not affect T-cell subset distribution significantly. We conclude that these hallmarks of age-associated alterations to immune signatures are indeed observed in the general population in people infected with CMV and not those infected with a different persistent herpesvirus.
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General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism
More LessThe onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36–38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.
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Genomic characterization of a novel human adenovirus type 31 recombinant in the hexon gene
More LessA novel human recombinant adenovirus of species A (HAdV-A31 MZ) was isolated from a patient with acute gastroenteritis in Japan. The complete genome of HAdV-A31 strain MZ contains 33 776 bp. Analysis of the hexon gene of HAdV-A31 MZ indicated that its hexon sequence is the result of a genetic recombination between those of HAdV-A31 and a close relative to HAdV-A12. The recombination sites were found around the border of hypervariable loops 1 and 2 in the hexon gene, which are the most important determinants for virus neutralization. Loops 1 and 2 of this virus were genetically related to HAdV-A12, whereas all other parts of the genome were highly similar to HAdV-A31. In order to understand the evolution of adenoviruses correctly and to avoid misidentification of HAdV types, we recommend characterizing not only the hexon gene, but also the penton base and fiber genes.
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Risk of seropositivity to multiple oncogenic human papillomavirus types among human immunodeficiency virus-positive and -negative Ugandan women
To understand the prospects for human papillomavirus (HPV) mass vaccination in the setting of a developing country, we studied the co-occurrence of seropositivity to multiple high-risk (hr) HPV types among HIV-positive and HIV-negative Ugandan women. Our seroepidemiological study was conducted among 2053 women attending antenatal clinics. Sera were analysed for antibodies to eight hrHPV types of the α-7 (18/45) and α-9 (16/31/33/35/52/58) species of HPV by using a multiplex serology assay. Our results show that seropositivity for greater than one hrHPV type was as common (18 %) as for a single type (18 %). HIV-positive women had higher HPV16, HPV18 and HPV45 seroprevalences than HIV-negative women. In multivariate logistic regression analysis, age (>30 years) and level of education (secondary school and above) reduced the risk, whereas parity (>5) and HIV-positivity increased the risk for multiple hrHPV seropositivity. However, in stepwise logistic regression analyses, HIV-status remained the only independent, stand-alone risk factor [odds ratio (OR) 1.7, 95 % confidence interval (CI) 1.0–2.8). On the other hand, the risk of HPV16 or HPV18 seropositive women, as compared to HPV16 or HPV18 seronegative women, for being seropositive to other hrHPV types was not significantly different when they were grouped by HIV-status (ORHPV16/HIV+ 12, 95 % CI 4.5–32 versus ORHPV16/HIV− 22, 95 % CI 15–31 and ORHPV18/HIV+ 58, 95 % CI 14–242 versus ORHPV18/HIV− 45, 95 % CI 31–65). In conclusion, seropositivity to HPV16, HPV18 and to non-vaccine hrHPV types is common in Ugandan women, suggesting that there is little natural cross-protective immunity between the types. HIV-positivity was an independent, stand-alone, albeit moderate risk factor for multiple hrHPV seropositivity. HPV mass vaccination may be the most appropriate method in the fight against cervical cancer in the Ugandan population.
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Human papillomavirus type-specific risk of cervical cancer in a population with high human immunodeficiency virus prevalence: case–control study
There are limited data on human papillomavirus (HPV) type-specific cervical cancer risk among human immunodeficiency virus (HIV)-positive women. Previous studies have suggested that HPV 16 would be relatively less important as a causative agent among HIV-positive compared with HIV-negative women. This study investigates HPV type-specific cervical cancer risk in a population in which HIV is endemic. At the Central Hospital, Maputo, Mozambique, 221 cervical cancer cases and 203 hospital-based controls were consecutively enrolled. HPV typing from cervical samples, HIV testing and recording of socio-demographic factors were performed. Logistic regression modelling was used to assess HPV type-specific risk and effect modification between HIV and HPV infection. Infection with HPV 16, 18 and ‘high-risk non-HPV 16/18 types’ (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) was associated with cervical cancer in both crude and adjusted analyses. HPV 16 and 18 were the most common types detected in cancer biopsies among both HIV-negative and HIV-positive women. There was no significant evidence of effect modification between any HPV type and HIV infection, and there were no significant differences in the HPV type-specific prevalence when cervical cancers among HIV-positive and HIV-negative women were compared. Within the limitations of the study, the relative importance of different HPV types in cervical carcinogenesis appears not to be modified greatly by HIV infection, suggesting that HPV vaccines might not need to be type-specifically modified to be suitable for populations where HIV is endemic.
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The large Rep protein of adeno-associated virus type 2 is polyubiquitinated
More LessFive adenovirus (Ad) gene products are required for efficient replication of co-infecting adeno-associated virus (AAV); however, the combined net enhancement by these factors is composed of both positive and negative effects. Similar to previous results with AAV Rep52, AAV2 large Rep was targeted for ubiquitination and degradation by the Ad E4orf6/E1b 55 kDa, cullin 5-containing, E3-ubiquitin ligase. Additionally, large Rep was targeted for ubiquitination via extension of ubiquitin lysine K48 and K63 both in the presence and absence of E4orf6.
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Comparative analysis of a highly variable region within the genomes of Spodoptera frugiperda ascovirus 1d (SfAV-1d) and SfAV-1a
More LessThe recently discovered ascoviruses have a worldwide distribution. Here we report a new member of the family Ascoviridae, Spodoptera frugiperda ascovirus 1d (SfAV-1d) with a variable region in the genome. Restriction fragment length polymorphism, Southern hybridization and genome sequencing analyses confirmed that SfAV-1d and the earlier reported SfAV-1a are closely related but are not identical. The genome size of SfAV-1d is approximately 100 kbp, which is about 57 kbp smaller than SfAV-1a. The SfAV-1d genome has a major deletion of 14 kbp that corresponds to one of the inverted repeat (IR) regions of SfAV-1a. Cloning and sequencing revealed that the region flanking the deletion within the SfAV-1d genome is highly variable. In all the variants of this region, the whole IR region is missing, with 88.2 % of the variants missing part of or the whole adjacent SfAV-1a ORF71, 94.1 % missing part of or the whole of adjacent ORF72 and 64.6 % missing part of or the whole of ORF73.
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- RNA viruses
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Identification of heat-shock protein 90 beta in Japanese encephalitis virus-induced secretion proteins
More LessFive host cellular proteins were identified in the secretion medium from Japanese encephalitis virus (JEV)-infected baby hamster kidney-21 (BHK-21) cells, including three molecular chaperones: Hsp70, GRP78 and Hsp90. Hsp90 isoforms were characterized further. Hsp90α was observed to be retained inside the nuclei, whereas Hsp90β associated with virus particles during assembly and was released into the secretion medium upon JEV infection. The association of Hsp90β and viral E protein was demonstrated by using sucrose-density fractionation and Western blot analysis. Moreover, JEV viral RNA replication was not affected by treatment with geldanamycin, an Hsp90 inhibitor, but impaired virus infectivity that was determined by a plaque-forming assay. Our results show that Hsp90β, not Hsp90α, is present in the JEV-induced secretion medium and is required for JEV infectivity in BHK-21 cells.
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Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate
The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.
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Flavivirus-induced antibody cross-reactivity
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.
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Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses
Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.
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Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions
We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.
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Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002–2009
More LessFoot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. Three different serotypes of the virus, namely O, A and Asia-1, are responsible for the outbreaks of this disease in these countries. In the present study, the nucleotide-coding sequences for the VP1 capsid protein (69 samples) or for all four capsid proteins (P1, seven representative samples) of the serotype A FMD viruses circulating in Pakistan and Afghanistan were determined. Phylogenetic analysis of the foot-and-mouth disease virus (FMDV) VP1-coding sequences from these countries collected between 2002 and 2009 revealed the presence of at least four lineages within two distinct genotypes, all belonging to the Asia topotype, within serotype A. The predominant lineage observed was A-Iran05 but three other lineages (a new one is named here A-Pak09) were also identified. The A-Iran05 lineage is still evolving as revealed by the presence of seven distinct variants, the dominant being the A-Iran05AFG-07 and A-Iran05BAR-08 sublineages. The rate of evolution of the A-Iran05 lineage was found to be about 1.2×10−2 substitutions per nucleotide per year. This high rate of change is consistent with the rapid appearance of new variants of FMDV serotype A in the region. The A22/Iraq FMDV vaccine is antigenically distinct from the A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino acid substitutions, exposed on the virus surface, which may explain this antigenic difference.
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New insights into RNA packaging in porcine reproductive and respiratory syndrome virus
More LessWhile identifying whether the smallest packaged heteroclite subgenomic RNA (S9) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a packaging signal, we found that S9 was capable of binding to the basic amino acid-rich domain (synthetic peptide of aa 34–53) of the packaging protein (N). In addition, by using truncations at the 5′ and 3′ ends of S9, a minimal binding region of 35 nt was found to be essential for binding to both the synthetic peptide and to the full-length N protein. Furthermore, by using cell-culture experiments, we found that S9 was capable of packaging non-viral RNA sequence into PRRSV particles and that the 35 nt region was essential for this activity. Taken together, our data suggest that this 35 nt region might be an important element for packaging PRRSV genomic RNA into virus particles.
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Outbreak of swine influenza in Argentina reveals a non-contemporary human H3N2 virus highly transmissible among pigs
Sporadic outbreaks of human H3N2 influenza A virus (IAV) infections in swine populations have been reported in Asia, Europe and North America since 1970. In South America, serological surveys in pigs indicate that IAVs of the H3 and H1 subtypes are currently in circulation; however, neither virus isolation nor characterization has been reported. In November 2008, an outbreak of respiratory disease in pigs consistent with swine influenza virus (SIV) infection was detected in Argentina. The current study describes the clinical epidemiology, pathology, and molecular and biological characteristics of the virus. Phylogenetic analysis revealed that the virus isolate shared nucleotide identities of 96–98 % with H3N2 IAVs that circulated in humans from 2000 to 2003. Antigenically, sera from experimentally inoculated animals cross-reacted mainly with non-contemporary human-origin H3N2 influenza viruses. In an experimental infection in a commercial swine breed, the virus was of low virulence but was transmitted efficiently to contact pigs and caused severe disease when an infected animal acquired a secondary bacterial infection. This is the first report of a wholly human H3N2 IAV associated with clinical disease in pigs in South America. These studies highlight the importance of two-way transmission of IAVs and SIVs between pigs and humans, and call for enhanced influenza surveillance in the pig population worldwide.
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Replication-incompetent influenza A viruses that stably express a foreign gene
A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.
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Junín virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2α phosphorylation
More LessStress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.
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Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles
The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.
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Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase
More LessThe viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.
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Modification of the trypsin cleavage site of rotavirus VP4 to a furin-sensitive form does not enhance replication efficiency
The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestines in vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.
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Genetic divergence and classification of non-structural protein 1 among porcine rotaviruses of species B
More LessPorcine rotavirus B (RVB) has frequently been detected in diarrhoea of suckling and weaned pigs. Moreover, epidemiological studies using ELISA have demonstrated high antibody prevalence in sera from sows, indicating that RVB infections are widespread. Because it is difficult to propagate RVBs serially in cell culture, genetic analysis of RNA segments of porcine RVBs other than those encoding VP7 and NSP2 has been scarcely performed. We conducted sequence and phylogenetic analyses focusing on non-structural protein 1 (NSP1), using 15 porcine RVB strains isolated from diarrhoeic faeces collected around Japan. Sequence analysis showed that the porcine NSP1 gene contains two overlapping ORFs. Especially, peptide 2 of NSP1 retains highly conserved cysteine and histidine residues among RVBs. Comparison of NSP1 nucleotide and deduced amino acid sequences from porcine RVB strains demonstrated low identities to those from other RVB strains. Phylogenetic analysis of RVB NSP1 revealed the presence of murine, human, ovine, bovine and porcine clusters. Furthermore, the NSP1 genes of porcine RVBs were divided into three genotypes, suggesting the possibility that porcine species might be an original host of RVB infection. Of nine strains common to those used in our previous study, only one strain was classified into a different genotype from the others in the analysis of VP7, in contrast to the analysis of NSP1, where all belonged to the same cluster. This fact suggests the occurrence of gene reassortment among porcine RVBs. These findings should provide more beneficent information to understand the evolution and functions of RVBs.
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Evolutionary relationship of the L- and M-class genome segments of bat-borne fusogenic orthoreoviruses in Malaysia and Australia
More LessWe previously described three new Malaysian orthoreoviruses designated Pulau virus, Melaka virus and Kampar virus. Melaka and Kampar viruses were shown to cause respiratory disease in humans. These viruses, together with Nelson Bay virus, isolated from Australian bats, are tentatively classified as different strains within the species Pteropine orthoreovirus (PRV), formerly known as Nelson Bay orthoreovirus, based on the small (S) genome segments. Here we report the sequences of the large (L) and medium (M) segments, thus completing the whole-genome characterization of the four PRVs. All L and M segments were highly conserved in size and sequence. Conserved functional motifs previously identified in other orthoreovirus gene products were also found in the deduced proteins encoded by the cognate segments of these viruses. Detailed sequence analysis identified two genetic lineages divided into the Australian and Malaysian PRVs, and potential genetic reassortment among the M and S segments of the three Malaysian viruses.
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Analysis of the human immunodeficiency virus type 1 M group Vpu domains involved in antagonizing tetherin
More LessZoonosis of chimpanzee simian immunodeficiency virus cpz to humans has given rise to both pandemic (M) and non-pandemic (O, N and P) groups of human immunodeficiency virus type-1 (HIV). These lentiviruses encode accessory proteins, including Vpu, which has been shown to reduce CD4 levels on the cell surface, as well as increase virion release from the cell by antagonizing tetherin (CD317, BST2). Here, we confirm that O group Vpus (Ca9 and BCF06) are unable to counteract tetherin or downregulate the protein from the cell surface, although they are still able to reduce cell-surface CD4 levels. We hypothesize that this inability to antagonize tetherin may have contributed to O group viruses failing to achieve pandemic levels of human-to-human transmission. Characterization of chimeric O/M group Vpus and Vpu mutants demonstrate that the Vpu–tetherin interaction is complex, involving several domains. We identify specific residues within the transmembrane proximal region that, along with the transmembrane domain, are crucial for tetherin counteraction and enhanced virion release. We have also shown that the critical domains are responsible for the localization of M group Vpu to the trans-Golgi network, where it relocalizes tetherin to counteract its function. This work sheds light on the acquisition of anti-tetherin activity and the molecular details of pandemic HIV infection in humans.
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