[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.
Preview · Article · Nov 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA(-) mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA(-) mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.
No preview · Article · Nov 2009 · Microbial Pathogenesis
[Show abstract][Hide abstract] ABSTRACT: Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was used as a vector to express the hemagglutinin (HA) protein of highly pathogenic H5N1 avian influenza virus, A/Chicken/Hubei/327/2004 (HB/327). The resultant recombinant baculovirus (BV-G-HA) mediated gene delivery and HA expression efficiently in mammalian cells. Mice immunized with 1 x 10(9)PFU of BV-G-HA developed significantly higher levels of H5-specific antibodies and cellular immunity than those that received 100 microg of DNA vaccines expressing HA, and were completely protected from lethal challenge with HB/327. Different vaccination doses were further tested in chickens, and these experiments demonstrated that 1 x 10(8)PFU of BV-G-HA offered complete protection from challenge with 100 LD(50) of HB/327. These data indicate that the pseudotype baculovirus-mediated vaccine could be utilized as an alternative strategy against the pandemic spread of H5N1 influenza virus.
Full-text · Article · Jun 2009 · Molecular Immunology
[Show abstract][Hide abstract] ABSTRACT: A sandwich ELISA test using AIV H5 subtype specific monoclonal antibody (clone 2H4) to an epitope of hemagglutinin protein has been developed. The monoclonal antibody was used to capture the antigen from clinical samples (swabs and tissues). Captured antigens from clinical samples were detected using polyclonal sera, purified AIV H5N1 particles were titrated in the sandwich ELISA and the limit of detection was determined to be approximately 1.0 ng of influenza viral protein in virus preparations. Fifteen AIV strains of H1-H15 subtypes and some other pathogens were tested by this system, and the test is specific to H5 subtype viruses as it failed to detect other AIV subtype viruses and other pathogens. Varieties of clinical samples originating from laboratory experiments (n=382) and from fields (n=288) were employed to test the efficacy of DAS-ELISA test. The test compared very well with the traditional method for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs. In comparison to virus isolation the sensitivity and specificity of sandwich ELISA were found to be 98.6% and 97.6% respectively. In addition, the DAS-ELISA was used to test samples of experimentally infected birds and clinical samples obtained from central China in 2005. The assay proved to be sensitive and specific for the rapid detection of AIV H5 subtype virus form the tissues and swabs in infected animals.
No preview · Article · May 2009 · Veterinary Microbiology
[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: 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: 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.
[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.
No preview · Article · Oct 2008 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is a powerful tool to silence gene expression. Small interfering RNA (siRNA)-induced RNA degradation has been recently used as an antivirus agent to inhibit specific virus replication. Here, we showed that several siRNAs specific for conserved regions of influenza virus matrix (M2) and nucleocapsid protein (NP) genes could effectively inhibit expression of the corresponding viral protein. We also evaluated the antiviral potential of these siRNAs targeting M2 and NP of H5N1 avian influenza virus (AIV), which are essential to viral replication. We investigated the inhibitory effect of M2-specific siRNAs and NP-specific siRNAs on influenza A virus (H5N1, H1N1 and H9N2) replication in Madin-Darby canine kidney (MDCK) cells and BALB/c mice. The results showed that treatment with these siRNAs could specifically inhibit influenza A virus replication in MDCK cells (0.51-1.63 TCID(50) reduction in virus titers), and delivery of pS-M48 and pS-NP1383 significantly reduced lung virus titers in the infected mice (16-50-fold reduction in lung virus titers) and partially protected the mice from lethal influenza virus challenge (a survival rate of 4/8 for H1N1 virus-infected mice and 2/8 for H5N1 virus infected mice). Moreover, the treatment of pS-M48 and pS-NP1383 could suppress replication of different subtypes of influenza A viruses, including a H5N1 highly pathogenic avian isolate strain. The results provided a basis for further development of siRNA for prophylaxis and therapy of influenza virus infection in humans and animals.
Full-text · Article · Dec 2007 · Antiviral Research
[Show abstract][Hide abstract] ABSTRACT: In 2004, 3 and 4 strains of avian influenza virus (subtype H5N1) were isolated from waterfowl and chickens, respectively, in central People's Republic of China. Viral replication and pathogenicity were evaluated in chickens, quails, pigeons, and mice. We analyzed the sequences of the hemagglutinin and neuraminidase genes of the isolates and found broad diversity among them.
Preview · Article · Jun 2007 · Emerging infectious diseases
[Show abstract][Hide abstract] ABSTRACT: A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as HIN1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test,and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.
Preview · Article · Mar 2007 · Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
[Show abstract][Hide abstract] ABSTRACT: BALB/c mice were immunized with formalin-treated influenza A/CK/Hubei/327/2004 virus. Six monoclonal antibodies specific to HA were selected, designed 1H8, 1D11, 2B7, 2C9, 2H4 and 4C9, respectively. The six Mabs probed linear epitopes by western blot assays. In ELISA additivity assays, the low additivity indexes (< or =28.3) of each pair Mabs indicated that the epitopes recognized by the six Mabs were located on the globular head of HA1. The neutralization activity of anti-HA1 Mabs and chicken polyclonal sera to various AIV H5N1 strains from different hosts was followed by virus neutralization with MDCK cells. All Mabs except 2C9 and chicken polyclonal serum showed highest neutralizing activity to lowly virulent A/Duck/XF/XFY/2004 from different phylogenetic lineage, and lowest neutralization efficiency to highly virulent A/CK/XF/XFJ/2004. For the other two highly virulent viruses, 1D11, 2H4, 4C9 and chicken polyclonal sera had higher neutralization to A/Goose/ZF/ZFE/2004 than A/CK/Hubei/327/2004, and 1H8 and 2B7 had considerable level of neutralizing efficiency to them. These findings suggested that the neutralizing antibodies showed lower neutralization efficiency to highly virulent virus strains than lowly virulent virus strains and strong cross-neutralizing reaction between virus strains located in different phylogenetic lineages. Moreover, the neutralizing Mabs could more efficiently neutralize AIV H5N1 strains from the natural hosts generally, such as waterfowl.
No preview · Article · Feb 2007 · Molecular Immunology
[Show abstract][Hide abstract] ABSTRACT: A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000-2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000-2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.
[Show abstract][Hide abstract] ABSTRACT: A latex agglutination test (LAT) based on polystyrene beads sensitized with inactivated avian influenza virus H5N1 particles
was developed. Compared with the hemagglutination inhibition test, the sensitivity and specificity of the LAT were 88.8 and
97.6%, respectively, in detecting 830 serum samples from vaccinated chickens. The test has application potential in field
Preview · Article · May 2005 · Journal of Clinical Microbiology