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ABSTRACT: Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4(+) and CD8(+) cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-γ, and blocking IFN-γ also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-γ reduced antibody titer, confirming that IFN-γ plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-γ-secreting cells might, in some situations, be less protective than those that do not.
Proceedings of the National Academy of Sciences 03/2013; · 9.68 Impact Factor
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ABSTRACT: Background. Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract illness (LRI) in children. Several promising live-attenuated RSV vaccines are in development. Defining additional markers of attenuation could enhance clinical trials.Methods. We used clinical data, virologic data, and nasal wash (NW) specimens from 20 RSV-naïve children enrolled in studies of 4 live-attenuated RSV vaccines. Seven received minimally-attenuated cpts248/955 or cpts530/1009 (Group 1), 6 received moderately-attenuated cpts248/404 (Group 2), and 7 received highly-attenuated rA2cp248/404/1030/ΔSH (Group 3). NW specimens were tested for cytokines and chemokines via electrochemiluminescence biosensor assay.Results. Group 1 exhibited one instance of LRI and significantly higher rates of fever than Groups 2, or 3; there were no significant differences in peak titers of vaccine virus. In contrast, levels of IFN-γ, IL-1β, IL-2, IL-6, and IL-13 were significantly greater in NW specimens from Group 1 compared to Group 3. Maximum increases in most cytokines occurred after peak viral replication but coincided with clinical illness.Conclusions. Substantial increases in proinflammatory, anti-inflammatory, Th1, Th2, and regulatory cytokines were detected in children administered minimally-attenuated live RSV vaccines, but not in children who received highly-attenuated vaccines. NW cytokines may be a useful biomarker of attenuation for live RSV vaccines.
The Journal of Infectious Diseases 03/2013; · 6.41 Impact Factor
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ABSTRACT: To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks.
PLoS ONE 01/2013; 8(1):e50598. · 4.09 Impact Factor
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Manoharan Parthiban,
Manimaran Kaliyaperumal,
Sa Xiao,
Baibaswata Nayak,
Anandan Paldurai,
Shin-Hee Kim,
Brian S Ladman,
Lauren A Preskenis,
Jack Gelb, Peter L Collins,
Siba K Samal
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ABSTRACT: An avian paramyxovirus type 4 (APMV-4) was isolated from a duck in Delaware in 2010. Its genome is 15,048 nucleotides (nt) long, which is shorter by 6 nt than those for all previously reported strains. Phylogenetic analysis revealed that this strain formed a separate cluster within APMV-4 strains.
Genome announcements. 01/2013; 1(1).
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ABSTRACT: The complete genome sequence was determined for a highly virulent Newcastle disease virus strain from vaccinated chicken farms in Mexico during outbreaks in 2010. On the basis of phylogenetic analysis this strain was classified into genotype V in the class II cluster that was closely related to Mexican strains that appeared in 2004-2006.
Genome announcements. 01/2013; 1(1).
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ABSTRACT: Newcastle disease (ND) is a deadly avian disease worldwide. In Africa, ND is enzootic and causes large economic losses, but little is known about the Newcastle disease virus (NDV) strains circulating in African countries. In this study, 27 NDV isolates from apparently healthy chickens in live bird markets of West African countries Benin and Togo during 2009 were characterized. All isolates had polybasic F protein cleavage sites and were shown to be highly virulent in standard pathogenicity assays. Infection of two-week-old chickens with two of the isolates resulted in 100% mortality within four days. Phylogenetic analysis of the 27 isolates based on a partial F gene sequence identified three clusters: one containing all the isolates from Togo and one from Benin (cluster 2), one containing most isolates from Benin (cluster 3), and an outlier isolate from Benin (cluster 1). All the three clusters are related to genotype VII strains of NDV. In addition, the cluster of viruses from Togo contained a recently-identified 6-nucleotide insert between the HN and L genes in a complete genome of NDV isolate from this geographical region. Multiple strains that include this novel element suggest local emergence of a new genome-length class. These results reveal genetic diversity within and among local NDV populations in Africa. Sequence analysis showed that the F and HN proteins of six West African isolates share 83.2 to 86.6% and 86.5 to 87.9% identity with vaccine strain LaSota, indicative of considerable diversity. A vaccine efficacy study showed that the LaSota vaccine protected birds from morbidity and mortality but did not prevent shedding of West African challenge viruses.
Journal of clinical microbiology 12/2012; · 4.16 Impact Factor
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ABSTRACT: Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. In previous work to develop point mutations in RSV with improved genetic stability, we observed that an attenuating mutation at amino acid position 1321 in the L polymerase protein was subject to de-attenuation by a spontaneous second-site compensatory mutation at position 1313 (Luongo et al., J. Virol., 2012). In the present study, we found that deletion of position 1313 (Δ1313), irrespective of the presence of an attenuating mutation at position 1321, provided a new attenuating mutation. RSV bearing Δ1313 replicated in cell culture as efficiently as wild type virus at 32°C, was restricted for replication at 37°C, and was restricted 50-fold and 150-fold in the upper and lower respiratory tract, respectively, of mice. We combined the Δ1313 deletion with the previously described, attenuating NS2 gene deletion (ΔNS2) to produce the recombinant live-attenuated RSV vaccine candidate ΔNS2/Δ1313. During in vitro stress tests involving serial passage at incrementally increasing temperatures, a second-site compensatory mutation was detected in close proximity of Δ1313, namely I1314T. This site was genetically and phenotypically stabilized by an I1314L substitution. Combination of I1314L with ΔNS2/Δ1313 yielded a virus, ΔNS2/Δ1313/1314L, with genetic stability at physiological temperature. This stabilized vaccine candidate was moderately temperature-sensitive and had a level of restriction in chimpanzees comparable to that of MEDI-559, a promising RSV vaccine candidate that presently is in clinical trials but lacks stabilized attenuating mutations. The level of attenuation and genetic stability identify ΔNS2/Δ1313/1314L as a promising candidate for evaluation in pediatric phase I studies.
Journal of Virology 12/2012; · 5.40 Impact Factor
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ABSTRACT: Avian paramyxoviruses (APMVs) belong to the genus Avulavirus in the family Paramyxoviridae and include at least nine serotypes, APMV-1 to -9, as well as two additional provisional serotypes. Newcastle disease virus (NDV), which comprises APMV-1, is the most extensively studied APMV because it is an important poultry pathogen. A moderate level of antigenic and genetic diversity is recognized for APMV-1 isolates, but our knowledge of the antigenic and genetic diversity of the other APMV serotypes is limited. APMV-4 is frequently isolated from waterfowl around the world. To date complete genome sequences of APMV-4 are available for only strains, which were isolated from ducks in Hong Kong, Korea and Belgium over a period of 37 years. We have carried out genome sequencing from the nucleocapsid (N) gene-end signal to the polymerase (L) gene-start signal of five APMV-4 strains recently isolated from Italy. Each of the eight APMV-4 strains has the same F protein cleavage site, DIQPR↓F. They also share a high level of nucleotide and amino acid sequence identity: for example, the F and HN glycoproteins have greater than 97% sequence identity between the various strains. Thus, comparison of these eight strains of APMV-4 did not provide evidence of substantial diversity, in contrast to similar studies with APMV-2, -3, and -6, in which the F and HN glycoproteins exhibited up to 20-30% amino acid sequence variation within a subgroup. Reciprocal cross-HI assay using post infection chicken sera also failed to detect significant antigenic variation among the available APMV-4 strains.
Virus Research 11/2012; · 2.94 Impact Factor
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ABSTRACT: Respiratory syncytial virus (RSV) forms cytoplasmic inclusion bodies (IBs) that are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. The present study found that IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the post-translational addition of OGN to protein targets to regulate cellular processes including signal transduction, transcription, translation, and the stress response. The virus-induced sequestration of p38-P in IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: oxidative stress resulting from arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN-addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection.
Journal of Virology 11/2012; · 5.40 Impact Factor
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Shin-Hee Kim,
Subhashree Nayak,
Anandan Paldurai,
Baibaswata Nayak,
Arthur Samuel,
Gilbert L Aplogan,
Kodzo A Awoume,
Richard J Webby,
Mariette F Ducatez, Peter L Collins,
Siba K Samal
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ABSTRACT: The complete genome sequence of an African Newcastle disease virus (NDV) strain isolated from a chicken in Togo in 2009 was determined. The genome is 15,198 nucleotides (nt) in length and is classified in genotype VII in the class II cluster. Compared to common vaccine strains, the African strain contains a previously described 6-nt insert in the downstream untranslated region of the N gene and a novel 6-nt insert in the HN-L intergenic region. Genome length differences are a marker of the natural history of NDV. This is the first description of a class II NDV strain with a genome of 15,198 nt and a 6-nt insert in the HN-L intergenic region. Sequence divergence relative to vaccine strains was substantial, likely contributes to outbreaks, and illustrates the continued evolution of new NDV strains in West Africa.
Journal of Virology 10/2012; 86(20):11394-5. · 5.40 Impact Factor
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ABSTRACT: Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.
Virology 09/2012; 433(2):320-8. · 3.35 Impact Factor
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ABSTRACT: The respiratory syncytial virus (RSV) G and F glycoproteins are the neutralization antigens, and G also is expressed in a soluble form (sG). Previously, sG was demonstrated to reduce the efficiency of RSV antibody-mediated neutralization by serving as an antigen decoy and to inhibit the antibody-mediated antiviral effects of Fc receptor-bearing leukocytes. The present study demonstrated that effective antibody-mediated restriction in vivo, and the evasion of this restriction by sG, involves pulmonary macrophages and complement, but not neutrophils.
Journal of Virology 07/2012; 86(19):10880-4. · 5.40 Impact Factor
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ABSTRACT: Human respiratory syncytial virus (RSV) is the most important viral cause of serious pediatric respiratory illness worldwide. Currently, the most promising live-attenuated vaccine candidate is a temperature-sensitive (ts) cDNA-derived virus named rA2cp248/404/1030ΔSH, in reference to its set of attenuating mutations. In a previous clinical study, more than one-third of postvaccination nasal wash isolates exhibited partial loss of the ts phenotype. Most of this instability appeared to be due to reversion at a missense point mutation called 1030. This 1030 mutation is a single-nucleotide tyrosine-to-asparagine substitution at position 1321 (Y1321N) of the polymerase L protein that contributes to the ts and attenuation phenotypes of the vaccine candidate. The goals of the present study were to identify a reversion-resistant codon at position 1321 conferring a comparable level of attenuation and to use this to develop a genetically stable version of the vaccine virus. We modified wild-type (wt) RSV to insert each of the 20 possible amino acids at position 1321; 19 viruses were recoverable. We also investigated small deletions at or near this position, but these viruses were not recoverable. Phenotypic analysis identified alternative attenuating amino acids for position 1321. Several of these amino acids were predicted, based on the genetic code, to be refractory to deattenuation. Classical genetics, using temperature stress tests in vitro combined with nucleotide sequencing, confirmed this stability but identified a second site with a compensatory mutation at position 1313. It was possible to stabilize the 1313 site as well, providing a stable 1030 mutation. Further stress tests identified additional incidental mutations, but these did not reverse the ts/attenuation phenotype. An improved version of the vaccine candidate virus was constructed and validated in vitro by temperature stress tests and in vivo by evaluation of attenuation in seronegative chimpanzees. In addition to developing an improved version of this promising live-attenuated RSV vaccine candidate, this study demonstrated the propensity of an RNA virus to escape from attenuation but also showed that, through systematic analysis, genetics can be used to cut off the routes of escape.
Journal of Virology 07/2012; 86(19):10792-804. · 5.40 Impact Factor
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ABSTRACT: Human parainfluenza viruses (HPIVs) are a common cause of acute respiratory illness throughout life. Infants, children, and the immunocompromised are the most likely to develop severe disease. HPIV1 and HPIV2 are best known to cause croup while HPIV3 is a common cause of bronchiolitis and pneumonia. HPIVs replicate productively in respiratory epithelial cells and do not spread systemically unless the host is severely immunocompromised. Molecular studies have delineated how HPIVs evade and block cellular innate immune responses to permit efficient replication, local spread, and host-to-host transmission. Studies using ex vivo human airway epithelium have focused on virus tropism, cellular pathology and the epithelial inflammatory response, elucidating how events early in infection shape the adaptive immune response and disease outcome.
Current opinion in virology. 06/2012; 2(3):294-9.
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ABSTRACT: Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.
Journal of Virology 05/2012; 86(10):5969-70. · 5.40 Impact Factor
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ABSTRACT: Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.
Journal of Virology 03/2012; 86(10):5829-43. · 5.40 Impact Factor
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ABSTRACT: The avian paramyxoviruses (APMVs) belong to the genus Avulavirus of family Paramyxoviridae. The APMVs are classified into nine serotypes on the basis of hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays, although some serologic cross-reaction exists. Newcastle disease virus (NDV), which constitutes serotype 1 (APMV-1), is an important pathogen of poultry, but the pathogenic potential of the other APMV serotypes is poorly understood. Although antibodies to APMV -2 to -9 are prevalent in chickens, the effect of prior exposure to these serotypes on susceptibility to NDV infection and disease was not known. In the present study, chickens were immunized with APMV-2 to -9 by the oculo-nasal route and later were challenged by the same route with a highly virulent strain of NDV. Among APMV-2 to -9, only APMV-3 induced serum antibodies that cross-reacted significantly with NDV and had significant NDV-neutralizing activity in vitro. In mock-immunized chickens, challenge NDV replicated throughout the respiratory tract as well as in the brain, spleen, and enteric tract. In contrast, in APMV-3-immunized chickens, challenge NDV replication was restricted to the upper respiratory tract and trachea. Some of the other APMVs also induced partial restriction of challenge NDV replication: for example, challenge NDV was not detected in the brains of APMV-9-immunized chickens, and shedding from the respiratory tract was reduced in chickens immunized with APMV-8 and -9. All of the chickens immunized with APMV-3 survived the NDV challenge; with APMV-2, -7, -8, and -9 the percentage survival was 30%, 20%, 20%, and 52.5%, respectively; whereas none of the chickens immunized with APMV-4, -5, or -6 survived. These results show that prior infection of chickens with APMV-3 induced substantial protection against NDV challenge, whereas prior infection with APMV-2, -7, -8, and -9 can alter subsequent NDV infection. The induction of NDV-neutralizing antibodies was a marker for efficient protection, but partial protection also was observed in their absence.
Vaccine 03/2012; 30(12):2220-7. · 3.77 Impact Factor
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Marcus L Hastie,
Madeleine J Headlam,
Nirav B Patel,
Alexander A Bukreyev,
Ursula J Buchholz,
Keyur A Dave,
Emma L Norris,
Cassandra L Wright,
Kirsten M Spann, Peter L Collins,
Jeffrey J Gorman
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ABSTRACT: Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.
Molecular & Cellular Proteomics 02/2012; 11(5):108-27. · 7.40 Impact Factor
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ABSTRACT: We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.
Journal of Virology 01/2012; 86(7):3828-38. · 5.40 Impact Factor
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ABSTRACT: Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.
PLoS ONE 01/2012; 7(2):e28382. · 4.09 Impact Factor
