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1.  The contribution of PA-X to the virulence of pandemic 2009 H1N1 and highly pathogenic H5N1 avian influenza viruses 
Scientific Reports  2015;5:8262.
PA-X is a novel protein encoded by PA mRNA and is found to decrease the pathogenicity of pandemic 1918 H1N1 virus in mice. However, the importance of PA-X proteins in current epidemiologically important influenza A virus strains is not known. In this study, we report on the pathogenicity and pathological effects of PA-X deficient 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza H5N1 viruses. We found that loss of PA-X expression in pH1N1 and H5N1 viruses increased viral replication and apoptosis in A549 cells and increased virulence and host inflammatory response in mice. In addition, PA-X deficient pH1N1 and H5N1 viruses up-regulated PA mRNA and protein synthesis and increased viral polymerase activity. Loss of PA-X was also accompanied by accelerated nuclear accumulation of PA protein and reduced suppression of PA on non-viral protein expression. Our study highlights the effects of PA-X on the moderation of viral pathogenesis and pathogenicity.
doi:10.1038/srep08262
PMCID: PMC4317690  PMID: 25652161
2.  Cryptoporus volvatus Extract Inhibits Influenza Virus Replication In Vitro and In Vivo 
PLoS ONE  2014;9(12):e113604.
Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.
doi:10.1371/journal.pone.0113604
PMCID: PMC4249958  PMID: 25437846
3.  H9N2 influenza virus in China: a cause of concern 
Protein & Cell  2014;6(1):18-25.
ABSTRACT
The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
doi:10.1007/s13238-014-0111-7
PMCID: PMC4286136  PMID: 25384439
Influenza; Poultry; H9N2; Evolution; Virulence; Antigenic drift
4.  H9N2 influenza virus in China: a cause of concern 
Protein & Cell  2014;6(1):18-25.
ABSTRACT
The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
doi:10.1007/s13238-014-0111-7
PMCID: PMC4286136  PMID: 25384439
Influenza; Poultry; H9N2; Evolution; Virulence; Antigenic drift
5.  Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice 
Journal of Virology  2014;89(1):2-13.
ABSTRACT
The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.
IMPORTANCE To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.
doi:10.1128/JVI.02390-14
PMCID: PMC4301103  PMID: 25320305
6.  Reconstruction of the Gene Regulatory Network Involved in the Sonic Hedgehog Pathway with a Potential Role in Early Development of the Mouse Brain 
PLoS Computational Biology  2014;10(10):e1003884.
The Sonic hedgehog (Shh) signaling pathway is crucial for pattern formation in early central nervous system development. By systematically analyzing high-throughput in situ hybridization data of E11.5 mouse brain, we found that Shh and its receptor Ptch1 define two adjacent mutually exclusive gene expression domains: Shh+Ptch1− and Shh−Ptch1+. These two domains are associated respectively with Foxa2 and Gata3, two transcription factors that play key roles in specifying them. Gata3 ChIP-seq experiments and RNA-seq assays on Gata3-knockdown cells revealed that Gata3 up-regulates the genes that are enriched in the Shh−Ptch1+ domain. Important Gata3 targets include Slit2 and Slit3, which are involved in the process of axon guidance, as well as Slc18a1, Th and Qdpr, which are associated with neurotransmitter synthesis and release. By contrast, Foxa2 both up-regulates the genes expressed in the Shh+Ptch1− domain and down-regulates the genes characteristic of the Shh−Ptch1+ domain. From these and other data, we were able to reconstruct a gene regulatory network governing both domains. Our work provides the first genome-wide characterization of the gene regulatory network involved in the Shh pathway that underlies pattern formation in the early mouse brain.
Author Summary
Recent large-scale projects of high-throughput in situ hybridization (ISH) have generated a wealth of spatiotemporal information on gene expression patterns in the early mouse brain. We have developed a computational approach that combines publicly available high-throughput ISH data with our own experimental data to investigate gene regulation, involving signal molecules and transcription factors (TFs), during early brain development. The analysis indicates that two key TFs, Foxa2 and Gata3, play antagonizing roles in the formation of two mutually exclusive domains established by the Sonic hedgehog signaling pathway in the developing mouse brain. Further ChIP-seq and RNA-seq experiments support this hypothesis and have identified novel target genes of Gata3, including the axon guidance regulators Slit2 and Slit3 as well as three neurotransmitter-associated genes, Slc18a1, Th and Qdpr. The findings have allowed us to reconstruct the gene regulatory network brought into play by the Sonic hedgehog pathway that mediates early mouse brain development.
doi:10.1371/journal.pcbi.1003884
PMCID: PMC4191885  PMID: 25299227
7.  Naturally Occurring Mutations in the PA Gene Are Key Contributors to Increased Virulence of Pandemic H1N1/09 Influenza Virus in Mice 
Journal of Virology  2014;88(8):4600-4604.
We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus's 50% mouse lethal dose (LD50), by almost 1,000-fold.
doi:10.1128/JVI.03158-13
PMCID: PMC3993729  PMID: 24522908
8.  Comparative Virus Replication and Host Innate Responses in Human Cells Infected with Three Prevalent Clades (2.3.4, 2.3.2, and 7) of Highly Pathogenic Avian Influenza H5N1 Viruses 
Journal of Virology  2014;88(1):725-729.
Highly pathogenic avian influenza H5N1 virus clades 2.3.4, 2.3.2, and 7 are the dominant cocirculating H5N1 viruses in poultry in China. However, humans appear to be clinically susceptible mostly to the 2.3.4 virus clade. Here, we demonstrated that A549 cells and human macrophages infected with clade 2.3.4 viruses produced significantly more viruses than those infected with the other two clades. Likewise, clade 2.3.4-infected macrophages caused the most severe cellular damage and strongest proinflammatory response.
doi:10.1128/JVI.02510-13
PMCID: PMC3911739  PMID: 24131718
10.  Pathogenic Pseudorabies Virus, China, 2012 
Emerging Infectious Diseases  2014;20(1):102-104.
In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic.
doi:10.3201/eid2001.130531
PMCID: PMC3884716  PMID: 24377462
pseudorabies; highly pathogenic PRV; China; viruses; pigs; swine
12.  Amino Acid 316 of Hemagglutinin and the Neuraminidase Stalk Length Influence Virulence of H9N2 Influenza Virus in Chickens and Mice 
Journal of Virology  2013;87(5):2963-2968.
H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice.
doi:10.1128/JVI.02688-12
PMCID: PMC3571412  PMID: 23269805
13.  Cryptoporus volvatus Extract Inhibits Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) In Vitro and In Vivo 
PLoS ONE  2013;8(5):e63767.
Porcine reproductive and respiratory syndrome virus (PRRSV) is an important arterivirus that can cause significant losses in swine industry. At present, there are no adequate control strategies against PRRSV. Thus, there is an urgent need for new treatment regimens that have efficacious antiviral activity to compensate for vaccines. Cryptoporus volvatus commonly serves as an anti-infective agent in Tradational Chinese Medicines. In this report, we exploited whether the aqueous extract from the fruiting body of Cryptoporus volvatus had the potential to inhibit PRRSV infection. Our results showed that the extract significantly inhibited PRRSV infection by repressing virus entry, viral RNA expression, and possibly viral protein synthesis, cell-to-cell spread, and releasing of virus particles. However, it did not block PRRSV binding to cells. Further studies confirmed that the extract directly inhibited PRRSV RNA-dependent RNA polymerase (RdRp) activity, thus interfering with PRRSV RNA and protein synthesis. More importantly, the extract efficiently inhibited highly pathologic PRRSV (HP-PRRSV) infection in vivo, reduced virus load in serum, and increased the survival rate of pigs inoculated with HP-PRRSV strain. Collectively, our findings imply that the aqueous extract from the fruiting body of Cryptoporus volvatus has the potential to be used for anti-PRRSV therapies.
doi:10.1371/journal.pone.0063767
PMCID: PMC3660591  PMID: 23704937
14.  Risk Perceptions for Avian Influenza Virus Infection among Poultry Workers, China  
Emerging Infectious Diseases  2013;19(2):313-316.
To determine risk for avian influenza virus infection, we conducted serologic surveillance for H5 and H9 subtypes among poultry workers in Beijing, China, 2009–2010, and assessed workers’ understanding of avian influenza. We found that poultry workers had considerable risk for infection with H9 subtypes. Increasing their knowledge could prevent future infections.
doi:10.3201/eid1902.120251
PMCID: PMC3563274  PMID: 23343592
Avian influenza; influenza; viruses; poultry workers; serologic survey; knowledge; attitudes; practices
15.  A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the Pathogenicity and Neurovirulence of H5N1 Influenza Virus in Mice 
Journal of Virology  2012;86(12):6924-6931.
H5 influenza viruses containing a motif of multiple basic amino acids at the hemagglutinin (HA) cleavage site (HACS) are highly pathogenic in chicken but display different virulence phenotypes in mammals. Previous studies have shown that multiple basic amino acids of H5N1 influenza virus are a prerequisite for lethality in mice. However, it remains unclear which specific residue at the cleavage site affects the pathogenicity of H5N1 in mammals. A comprehensive genetic analysis of the H5N1 HACS showed that residues at P6 (position 325, by H3 numbering) were the most polymorphic, including serine (S), arginine (R), deletion (*), glycine (G), and isoleucine (I). To determine whether a single residue at P6 could affect virulence, we introduced different mutations at P6 of an avirulent clade 7 H5N1 strain, rg325G, by reverse genetics. Among the recombinant viruses, the rg325S virus showed the highest cleavage efficiency in vitro. All these viruses were highly pathogenic in chicken but exhibited different virulences in mice. The rg325S virus exhibited the highest pathogenicity in terms of unrestricted organ tropism and neurovirulence. Remarkably, the HA-325S substitution dramatically increased the pathogenicity of H5N1 viruses of other clades, including clades 2.2, 2.3.2, and 2.3.4, indicating that this residue impacts genetically divergent H5N1 viruses. An analysis of predicted structures containing these mutations showed that the cleavage site loop with 325S was the most exposed, which might be responsible for the efficient cleavage and high virulence. Our results demonstrate that an amino acid substitution at the P6 cleavage site alone could modulate the virulence of H5N1 in mice.
doi:10.1128/JVI.07142-11
PMCID: PMC3393526  PMID: 22496231
16.  Identification of swine influenza A virus and Stenotrophomonas maltophilia co-infection in Chinese pigs 
Virology Journal  2012;9:169.
Background
Influenza virus virulence can be exacerbated by bacterial co-infections. Swine influenza virus (SIV) infection together with some bacteria is found to enhance pathogenicity.
Methods
SIV-positive samples suspected of containing bacteria were used for bacterial isolation and identification. Antimicrobial susceptibility testing was performed by disc diffusion methods. To investigate the interaction of SIV and the bacteria in vitro, guinea pigs were used as mammalian hosts to determine the effect on viral susceptibility and transmissibility. Differences in viral titers between groups were compared using Student’s t-test.
Results
During surveillance for SIV in China from 2006 to 2009, seven isolates (24.14%) of 29 influenza A viruses were co-isolated with Stenotrophomonas maltophilia from nasal and tracheal swab samples of pigs. Antimicrobial susceptibility testing showed that the bacteria possessed a high level of resistance towards clinically used antibiotics. To investigate the interaction between these two microorganisms in influencing viral susceptibility and transmission in humans, guinea pigs were used as an infection model. Animals were inoculated with SIV or S. maltophilia alone or co-infected with SIV and S. maltophilia. The results showed that although no transmission among guinea pigs was observed, virus–bacteria co-infections resulted in higher virus titers in nasal washes and trachea and a longer virus shedding period.
Conclusions
This is the first report of influenza virus co-infection with S. maltophilia in the Chinese swine population. Increased replication of virus by co-infection with multidrug resistant bacteria might increase the infection rate of SIV in humans. The control of S. maltophilia in clinics will contribute to reducing the spread of SIV in pigs and humans.
doi:10.1186/1743-422X-9-169
PMCID: PMC3492169  PMID: 22913775
Swine influenza virus; S. maltophilia; Co-infections; Guinea pigs
17.  Mouse-Adapted H9N2 Influenza A Virus PB2 Protein M147L and E627K Mutations Are Critical for High Virulence 
PLoS ONE  2012;7(7):e40752.
H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.
doi:10.1371/journal.pone.0040752
PMCID: PMC3393695  PMID: 22808250
18.  Duck Hepatitis A Virus Possesses a Distinct Type IV Internal Ribosome Entry Site Element of Picornavirus 
Journal of Virology  2012;86(2):1129-1144.
Sequence analysis of duck hepatitis virus type 1 (DHV-1) led to its classification as the only member of a new genus, Avihepatovirus, of the family Picornaviridae, and so was renamed duck hepatitis A virus (DHAV). The 5′ untranslated region (5′ UTR) plays an important role in translation initiation and RNA synthesis of the picornavirus. Here, we provide evidence that the 651-nucleotide (nt)-long 5′ UTR of DHAV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and within BHK cells. Comparative sequence analysis showed that the 3′ part of the DHAV 5′ UTR is similar to the porcine teschovirus 1 (PTV-1) IRES in sequence and predicted secondary structure. Further mutational analyses of the predicted domain IIId, domain IIIe, and pseudoknot structure at the 3′ end of the DHAV IRES support our predicted secondary structure. However, unlike the case for the PTV-1 IRES element, analysis of various deletion mutants demonstrated that the optimally functional DHAV IRES element with a size of approximately 420 nt is larger than that of PTV-1 and contains other peripheral domains (Id and Ie) that do not exist within the type IV IRES elements. The domain Ie, however, could be removed without significant loss of activity. Surprisingly, like the hepatitis A virus (HAV) IRES element, the activity of DHAV IRES could be eliminated by expression of enterovirus 2A protease. These findings indicate that the DHAV IRES shares common features with type IV picornavirus IRES elements, whereas it exhibits significant differences from type IV IRESs. Therefore, we propose that DHAV possesses a distinct type IV IRES element of picornavirus.
doi:10.1128/JVI.00306-11
PMCID: PMC3255860  PMID: 22090106
19.  Acute Respiratory Distress Syndrome Induced by a Swine 2009 H1N1 Variant in Mice 
PLoS ONE  2012;7(1):e29347.
Background
Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus.
Methodology Principal Findings
Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8–10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines.
Conclusions/Significance
These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus.
doi:10.1371/journal.pone.0029347
PMCID: PMC3250439  PMID: 22235288
20.  Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice 
Virology Journal  2011;8:505.
Background
H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses.
Methods
To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.
Results
Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs.
Conclusion
Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.
doi:10.1186/1743-422X-8-505
PMCID: PMC3236014  PMID: 22050764
avian influenza virus; H9N2; reassortant; genotype; pathogenicity
21.  Characterization of an Artificial Swine-Origin Influenza Virus with the Same Gene Combination as H1N1/2009 Virus: A Genesis Clue of Pandemic Strain 
PLoS ONE  2011;6(7):e22091.
Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.
doi:10.1371/journal.pone.0022091
PMCID: PMC3143117  PMID: 21799774
22.  Modification of Nonstructural Protein 1 of Influenza A Virus by SUMO1 ▿ ‡  
Journal of Virology  2010;85(2):1086-1098.
Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.
doi:10.1128/JVI.00877-10
PMCID: PMC3020006  PMID: 21047957
23.  Molecular epidemiology of dengue viruses in southern China from 1978 to 2006 
Virology Journal  2011;8:322.
To investigate molecular epidemiology of dengue viruses (DENV) in southern China, a total of 14 dengue isolates were collected in southern China during each epidemic year between 1978 and 2006 and their full-length genome sequences were obtained by using RT-PCR method. The E gene sequences from additional 6 dengue fever patients in Guangzhou in 2006 were also obtained by using RT-PCR method. Combined with DENVs sequences published in GenBank, phylogenetic analysis and recombination analysis were performed. One hundred and twenty-five E gene sequences and 60 complete genome sequences published in the GenBank were also involved. Phylogenetic analysis showed that there was a wide genetic diversity of DENVs isolated in southern China. DENV-1 strains exist in almost all of the clades of genotype I and IV except the Asia 1 clade of genotype I; DENV-2 stains are grouped into four of the five genotypes except American genotype. DENV-4 strains are grouped into 2 genotypes (I and II). Phylogenetic analysis also showed that all DENV-4 isolates and two DENV-2 isolates were closely related to the prior isolates from neighboring Southeast Asia countries. The DENV-1 strain isolated during the 2006 epidemic is highly homologous to the strains isolated during the 2001 epidemic.
Recombination analysis showed no inter-serotype recombination, but 22 intra-serotype recombination events were found across the 32 complete genomes of all Chinese isolates. The study suggested that dengue fever epidemic in Southern China over the past 30 years presented two important modes, 1) imported-cases-induced endemic prevalence; 2) endogenous epidemic outbreak with natural epidemic focus. Recombination may play an important role in dengue virus evolution and adaptation.
doi:10.1186/1743-422X-8-322
PMCID: PMC3138434  PMID: 21703015
24.  Influenza A Virus Replication Induces Cell Cycle Arrest in G0/G1 Phase▿  
Journal of Virology  2010;84(24):12832-12840.
Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G0/G1-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Consistent with the G0/G1-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G1 into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G0/G1-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G0/G1 phase were observed compared to those in either unsynchronized cells or cells synchronized in the G2/M phase. G0/G1-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G0/G1-phase cell cycle arrest in infected cells.
doi:10.1128/JVI.01216-10
PMCID: PMC3004346  PMID: 20861262
25.  Guinea Pig Model for Evaluating the Potential Public Health Risk of Swine and Avian Influenza Viruses 
PLoS ONE  2010;5(11):e15537.
Background
The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.
Methodology/Principal Findings
We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.
Conclusions/Significance
We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.
doi:10.1371/journal.pone.0015537
PMCID: PMC2990763  PMID: 21124850

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