[show abstract][hide abstract] ABSTRACT: Since the emergence of the 2009 pandemic (H1N1) virus (2009/H1N1) in April 2009, cases of transmission from humans to pigs have been reported frequently. In our previous studies, four 2009/H1N1 variants were isolated from pigs. To better understand the phenotypic differences of the pig isolates compared with the human isolate, in this study mice were inoculated intranasally with different 2009/H1N1 viruses, and monitored for morbidity, mortality, and viral replication, cytokine production and pathological changes in the lungs. The results show that all isolates show effective replication in lungs, but varying in their ability to cause morbidity. In particular, the strains of A/swine/Nanchang/3/2010 (H1N1) and A/swine/Nanchang/F9/2010 (H1N1) show the greatest virulence with a persisting replication in lungs and high lethality for mice, compared with the human isolate A/Liaoning /14/2009 (H1N1), which shows low virulence in mice. Furthermore, the lethal strains could induce more severe lung pathological changes and higher production of cytokines than that of other strains at an early stage. Amino acid sequence analysis illustrates prominent differences in viral surface glycoproteins and polymerase subunits between pig isolates and human strains that might correlate with their phenotypic differences. These studies demonstrate that the 2009/H1N1 pig isolates exhibit heterogeneous infectivity and pathogencity in mice, and some strains possess an enhanced pathogenicity compared with the human isolate.
Veterinary Research 06/2013; 44(1):41. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: In 2009, two H1N2 influenza viruses were isolated from trachea swabs of pigs in Hubei in China. We compared these sequences with the other 18 complete genome sequences of swine H1N2 isolates from China during 2004 to 2010 and undertook extensive analysis of their evolutionary patterns. Six different genotypes - two reassortants between triple reassortant (TR) H3N2 and classical swine (CS) H1N1 virus, three reassortants between TR H1N2, Eurasian avian-like H1N1 swine virus and H9N2 swine virus, and one reassortant between H1N1, H3N2 human virus and CS H1N1 virus - were observed in these 20 swine H1N2 isolates. The TR H1N2 swine virus is the predominant genotype, and the two Hubei H1N2 isolates were located in this cluster. We also used a mouse model to examine the pathogenesis and inflammatory responses of the two isolates. The isolates replicated efficiently in the lung, and exhibited a strong inflammatory response, serious pathological changes and mortality in infected mice. Given the role that swine can play as putative "genetic mixing vessels" and the observed transmission of TR H1N2 in ferrets, H1N2 influenza surveillance in pigs should be increased to minimize the potential threat to public health.
Archives of Virology 04/2013; · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.
PLoS ONE 01/2013; 8(4):e61397. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The highly pathogenic avian influenza (HPAI) H5N1 virus is a highly virulent pathogen that causes respiratory diseases and death in humans and other animal species worldwide. Because influenza is an enveloped virus, the entry, assembly, and budding of virus particles are essential steps in the viral life cycle, and the virus relies on the participation of host cellular membrane proteins for all of these steps. Thus, we took a comparative membrane proteomics approach by using 2-DE coupled with MALDI-TOF/TOF MS to profile membrane proteins involved in H5N1 virus infection at 6, 12, and 24 h. Forty-two different proteins were found to vary on A549 cells due to H5N1 virus infection. Of these proteins, 57% were membrane or membrane-associated proteins. To further characterize the roles of novel identified proteins in virus propagation, the siRNA technology were applied and complement component C1q binding protein, annexin 2, prohibitin, peroxiredoxin 1 and heat shock protein 90-beta were successfully demonstrated to be contributed to viral propagation. In conclusion, the present study provides important new insight into understanding the roles of host membrane proteins in viral infection progress, and this insight is of particular importance for the development of novel therapeutic strategies.
Journal of Proteome Research 09/2012; · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006-2007 were investigated in the present study. RESULTS: Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006-2007). HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006-2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites. CONCLUSIONS: The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.
[show abstract][hide abstract] ABSTRACT: Streptococcus suis serotype 2 (SS2), a major swine pathogen and an emerging zoonotic agent, has greatly challenged global public health. The encoding proteins with unknown functions the bacterium encodes are an obstruction to studies of the pathogenesis. A novel surface protective antigen HP0197 is one of these proteins which have no sequence homology to any known protein. In the present study, the protein was determined to be involved in bacterial virulence through an evaluation of the isogenic mutant (Δhp0197) in both mice and pigs. The experimental infection also indicated that Δhp0197 could be cleared easily during infection, which could be attributed to the reduced thickness of the capsular polysaccharides (CPS) and the significantly reduced phagocytotic resistance. Microarrays-based comparative transcriptome analysis suggested that the suppressed expression of the operon responsible for CPS synthesis might be reversed by CcpA activity, which controlled global regulation of carbon catabolite through the binding of the CcpA and HPr-Ser-46-P to the catabolite-responsive elements (cre) of the target operons. The hypothesis was approved by the fact that the purified FLAG-tagged HPr from WT stain exhibited a higher binding activity to cre with CcpA compared to the Δhp0197 by the Electrophoretic Mobility Shift Assay, suggesting lower level of phosphorylation of the phosphocarrier protein HPr at residue Ser-46 (HPr-Ser-46P) in Δhp0197. These indicated that HP0197 could enhance CcpA activity to control the expression of genes involved in carbohydrate utilization and CPS synthesis, thus contributing to the virulence of S. suis.
PLoS ONE 01/2012; 7(11):e50987. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The H1N1/2009 influenza virus has the potential to cause a human pandemic, and sporadic cases of human-to-pig transmission have been reported. In this study, two influenza viruses were isolated from pigs. A phylogenetic analysis showed that the A/swine/NanChang/F9/2010(H1N1) (F9/10) strain shared a high degree of homology with the pandemic H1N1/2009 virus, and A/swine/GuangDong/34/2006 (H1N1) (34/06) strains was a classical swine influenza virus. A proteomic analysis was performed to investigate possible alterations of protein expression in porcine alveolar macrophage (PAM) cells infected by the F9/10 and 34/06 viruses over different time courses. Using 2-DE in association with MALDI-TOF MS/MS, we identified 13 up-regulated and 21 down-regulated protein spots, including cytoskeleton proteins, cellular signal transduction proteins, molecular biosynthesis proteins and heat shock proteins. The most significant changes in the infected cells were associated with molecular biosynthesis proteins and heat shock proteins. We analysed the biological characteristics of the F9/10 and 34/06 viruses in vivo and in vitro. The F9/10 virus showed greater pathogenicity than the 34/06 virus in PAM cells and mice. This study provides insights into the biologic characteristics, potential virulence alteration and cross-species transmission mechanisms of the pandemic H1N1/2009.
Journal of proteomics 12/2011; 75(6):1732-41. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: To the Editor: During March and early April 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico and the United States; this virus subsequently spread across the globe by human-to-human transmission at an unprecedented rate. Pandemic (H1N1) 2009 virus also affected pigs. On May 2, 2009, the Canadian Food Inspection Agency notified the World Organisation for Animal Health that the novel influenza A virus had been confirmed on a pig farm in Alberta, Canada. Infection of pigs with pandemic (H1N1) 2009 virus has been observed in multiple countries (1). In this study, we report transmission of pandemic (H1N1) 2009 virus from humans to pigs in the People's Republic of China.
[show abstract][hide abstract] ABSTRACT: As a mild, highly contagious, respiratory disease, swine influenza always damages the innate immune systems, and increases susceptibility to secondary infections which results in considerable morbidity and mortality in pigs. Nevertheless, the systematical host response of pigs to swine influenza virus infection remains largely unknown. To explore it, a time-course gene expression profiling was performed for comprehensive analysis of the global host response induced by H1N1 swine influenza virus in pigs.
At the early stage of H1N1 swine virus infection, pigs were suffering mild respiratory symptoms and pathological changes. A total of 268 porcine genes showing differential expression (DE) after inoculation were identified to compare with the controls on day 3 post infection (PID) (Fold change ≥ 2, p < 0.05). The DE genes were involved in many vital functional classes, mainly including signal transduction, immune response, inflammatory response, cell adhesion and cell-cell signalling. Noticeably, the genes associated with immune and inflammatory response showed highly overexpressed. Through the pathway analysis, the significant pathways mainly concerned with Cell adhesion molecules, Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway and MAPK signaling pathway, suggesting that the host took different strategies to activate these pathways so as to prevent virus infections at the early stage. However, on PID 7, the predominant function classes of DE genes included signal transduction, metabolism, transcription, development and transport. Furthermore, the most significant pathways switched to PPAR signaling pathway and complement and coagulation cascades, showing that the host might start to repair excessive tissue damage by anti-inflammatory functions. These results on PID 7 demonstrated beneficial turnover for host to prevent excessive inflammatory damage and recover the normal state by activating these clusters of genes.
This study shows how the target organ responds to H1N1 swine influenza virus infection in pigs. The observed gene expression profile could help to screen the potential host agents for reducing the prevalence of swine influenza virus and further understand the molecular pathogenesis associated with H1N1 infection in pigs.
[show abstract][hide abstract] ABSTRACT: During 2006-2009 influenza virus surveillance, three H3N2 viruses were isolated from ducks in Central China. Sequence and phylogenetic analyses revealed that most segments of these three isolates had high identity with H3N2 swine isolates in South China. However, for M, the three viruses, along with H1N1 swine isolates of North America, formed a cluster; for PB2, two of these isolates fell into the cluster of the H5N1 duck isolates, indicating a reassortment among H3N2, H1N1 swine viruses and H5N1 avian virus. The emergence of H3N2 virus with incorporation of an H5N1 virus gene raises new concerns about the generation of novel viruses that could affect humans.
Archives of Virology 02/2011; 156(6):1045-8. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: RAS, coded by ras proto-oncogenes, played an important role in signal transmission to regulate cell growth and differentiation. Host activation of RAS was significant for IFN-sensitive vaccinia virus (delE3L) or attenuate influenza virus in unallowable cells.
Huamn NRAS gene was activated by mutating in codon 61. Then the activation of NRAS was detected by western blot in MDCK cells. The delNS1 H5N1 influenza virus with deletion of NS1 eIF4GI binding domain was weak multiplication in MDCK cells. And the replication of delNS1 virus and expression of IFN-beta and IRF-3 were detected by Real-time PCR in MDCK cells infected with delNS1 virus. It was found that the delNS1 virus had a significant increase in MDCK cells when the NRAS was activated, and yet, expression of IRF-3 and IFN-beta were restrained.
The study demonstrated that activated NRAS played an important part for delNS1 virus replication in MDCK cells. Activated NRAS might be down-regulating the expression of antiviral cellular factors in delNS1 virus infected cells.
[show abstract][hide abstract] ABSTRACT: The 2009 pandemic H1N1 influenza virus encodes an NS1 protein with 11 amino acids (aa) truncation at the C-terminus. The C-terminal tail of influenza virus NS1 protein constitutes a nucleolar localization signal (NoLS) and is the binding domain of the cellular pre-mRNA processing protein, poly(A)-binding protein II (PABII). Here, our studies showed that the C-terminal-truncated NS1 of the 2009 pandemic virus was inefficient at blocking host gene expression, extension of the truncated NS1 to its full length increased the inhibition of host gene expression. Mechanistically, this increased inhibition of host gene expression by the full-length NS1 was not associated with nucleolar localization, but was due to the restoration of NS1's binding capacity to PABII. Furthermore, in vitro and in vivo characterization of two recombinant viruses encoding either the C-terminal 11-aa truncated or full-length NS1 of the 2009 pandemic virus showed that the C-terminal 11-aa truncation in NS1 did not significantly alter virus replication, but increased virus pathogenicity in mice.
PLoS ONE 01/2011; 6(10):e26175. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the effect of NS1 eIF4GI binding domain on virulence and pathogenicity of H5N1 influenza A virus, 5 recombinant H5N1 viruses encoding eIF4GI binding domain-truncated NS1 proteins and parental NS1 (NS1‐wt) were generated by an 8‐plasmid-based reverse genetics system. The results indicated that the recombinants with the addition of 5‐amino acid and the deletion position of 85-89 in NS1‐wt were attenuated in replication in vitro and in vivo, compared with the recombinant wild‐type virus rNS1‐wt, whereas the deletion position 85-94 or the entire eIF4GI binding domain in NS1‐wt displayed a significantly attenuated phenotype in chicken and mice. We also showed that the eIF4GI binding domain-truncated mutants were impaired in their ability to inhibit interferon production in vitro, and they did not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in Madin ‐Darby Canine Kidney cells, or in vivo in chickens and in a mouse model. Therefore, these attenuated NS1‐truncated viruses may have a great potential as live attenuated vaccine candidates against H5N1 influenza A virus.
The Journal of Infectious Diseases 11/2010; 202(9):1338-46. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: A certain H5N1 avian influenza virus has gained the ability to cause the classic central nervous system dysfunction in poultry and migratory birds. This study presents the proteomics analysis on the change of proteins to H5N1 avian influenza virus with neurovirulence infection in chicken brain tissue. By using 2-DE, coupled with MALDI-TOF MS/MS, we identified a set of differentially expressed cellular proteins, including 18 up-regulated proteins and 13 down-regulated proteins. The most significant changes were found in cytoskeleton proteins, proteins associated with the ubiquitin-proteasome pathway, and neural signal transduction proteins. Some identified proteins such as CRMP and SEP5 were found to participate in the pathogenesis progress of Parkinson's and Huntington's diseases, which also developed encephalitis accompanied with CNS dysfunction. The obtained data can provide insight into the virus-chicken brain tissue interaction and reveal the potential mechanism of the neuropathogenesis when the host was infected by the neurovirulent avian influenza virus.
Journal of Proteome Research 05/2010; 9(8):3789-98. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. As traditional inactive vaccine has obvious shortcomings, to identify protective antigens would undoubtedly contribute to the development of novel vaccines. HP0197 has been identified as immunogenic protein in the previous study but its protective efficacy was not clear. In the present study, the purified recombinant HP0197 protein could elicit a significant humoral antibody response and could confer significant protection against challenge with lethal dose of SS2 in mice and pigs. In addition, the hyperimmune sera against HP0197 could efficiently kill the bacteria in the opsonized phagocytosis test and conferred significant protection against SS2 infection in the experiment of passive immunization. The present study suggests with strong evidence that the identified protective antigen would be a novel and an effective vaccine candidate for SS2.
[show abstract][hide abstract] ABSTRACT: During 2004-2006 swine influenza virus surveillance, two strains of H3N8 influenza viruses were isolated from pigs in central China. Sequence and phylogenetic analyses of eight gene segments revealed that the two swine isolates were of equine origin and most closely related to European equine H3N8 influenza viruses from the early 1990s. Comparison of hemagglutinin (HA) amino acid sequences showed several important substitutions. One substitution caused the loss of a potential glycosylation site, and two substitutions, located at the cleavage site and adjacent to the receptor-binding pocket, respectively, had been reported previously in canine H3 HAs. This expansion of host range of equine H3N8 influenza viruses with mutations in the HA protein might raise the possibility of transmission of these viruses to humans.
Archives of Virology 05/2009; 154(5):887-90. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Based on the understanding that the analysis on unique short (US) region of duck enteritis virus (DEV) might contribute to the recognition of the molecular characterization and the evolution of DEV, in the study, a 5,121 bp fragment, which contained three genes encoding complete US10, unique short region open reading frame (SORF) 3, and US2 proteins, was amplified from the DEV (C-KCE) genome. The transcription orientation of the US10 and SORF3 was in a tail-to-tail way, and the SORF3 and US2 was the same. Potential core promoters and polyadenylation (poly(A)) sites were predicted for US10, SORF3, and US2 and further confirmed by polymerase chain reaction. Phylogenetic analysis for the three complete coding sequences showed that DEV was more closely related to avian herpesviruses, especially to Mardivirus, and should be classified to a separate genus of the Alphaherpesvirinae subfamily.
[show abstract][hide abstract] ABSTRACT: The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49(th) to 68(th) positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.
PLoS ONE 02/2009; 4(7):e6277. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this report, an H5N1 avian influenza virus, A/duck/Hubei/hangmei01/2006, which could lead to acute disease including neurovirulence and mortality in ducks, was isolated in brains of domestic ducks in spring of 2006. Molecular characterization of the genes revealed that this virus harbored the common characteristics of a highly pathogenic avian influenza (HPAI). Phylogenetic analyses demonstrated that this virus was a member of the Fujian-like virus sublineage. All eight genes except NA and PB2 had the closest genetic relatives to the human influenza virus A/China/GD01/2006. It might indicate that the virus A/duck/Hubei/hangmei01/2006 originated from southern China, resulting from the wild bird migration or poultry transportation, and indicate that more surveillance upon evolution and transmission of influenza viruses in ducks was urgent.
[show abstract][hide abstract] ABSTRACT: An H5N1 avian influenza virus (AIV) hemagglutinin (HA) protein pseudotyped lentivirus, HIV/H5-HA, was generated, characterized in vitro and evaluated for its ability to induce protective immunity against virulent wild type AIV in mice. The HIV/H5-HA virus was able to infect 293T, BHK, Vero, PK-15, MDCK cells but not IBRS-2 cells and therefore demonstrated cell tropism similar to the wild type AIV. HIV/H5-HA agglutinated chicken erythrocytes and cell entry was blocked by ammonium chloride, indicating that the process is pH-dependent. In mice, HIV/H5-HA immunization resulted in low levels of virus in the lungs, elicited high levels of AIV HA-specific antibody as indicated by the hemagglutination inhibition (HI) test, and the antibody induction was both earlier and with a higher titer than that induced by the inactivated AIV vaccine. These results confirmed the roles played by HA in AIV infection and immunogenicity and suggested that the pseudotyped lentivirus is a good model for studying the functions of AIV HA.
Journal of Virological Methods 10/2008; 154(1-2):99-103. · 1.90 Impact Factor