NS1: A Key Protein in the "Game" Between Influenza A Virus and Host in Innate Immunity.

Since the influenza pandemic occurred in 1918, people have recognized the perniciousness of this virus. It can cause mild to severe infections in animals and humans worldwide, with extremely high morbidity and mortality. Since the first day of human discovery of it, the "game" between the influenza virus and the host has never stopped.

[Construction and biological characteristics of H5N1 avian influenza viruses with different patterns of the glycosylation sites in HA protein].

The distribution of glycosylation sites in HA proteins was various among H5 subtype avian influenza viruses (AIVs), however, the role of glycosylation sites to the virus is still unclear. In this study, avian influenza H5N1 viruses with deletion of the glycosylation sites in HA were constructed and rescued by site direct mutation and reverse genetic method, and their biological characteristics and virulence were determined. The result showed that the mutants were confirmed to be corrected by HA gene sequencing and Western blot analysis.

[Genome sequencing and phylogenetic analysis of avian influenza viruses subtype H9N2].

Samples of chicken, duck, quail, and pigeon were collected from Jiangsu, Anhui, and Hebei in 2009-2011, and sixteen H9N2 subtype isolates of avian influenza virus (AIV) were identified. The eight full-length genes of 16 AIV isolates were amplified by RT-PCR and sequenced. Genome sequence analysis showed that the amino acid motif of cleavage sites in the HA gene was P-S-R/K-S-S-R, which was consistent with the characterization of the LPAIV, and the Leucine (L) at the amino acid position 226 in the HA genes of all isolates indicated the potential of binding with SAalpha, 2-6 receptor.

[Preparation and characterization of monoclonal antibodies specific for CjaA protein of Campylobacter jejuni].

Aim: Expression, purification of Campylobacter jejuni CjaA protein and development of monoclonal antibodies (mAbs) against this protein.

Methods: The C. jejuni cjaA gene was amplified and inserted into the expression plasmids, pGEX-6p-1 and pET30a (+).

Intranasal immunization with chitosan/pCAGGS-flaA nanoparticles inhibits Campylobacter jejuni in a White Leghorn model.

Campylobacter jejuni is the most common zoonotic bacterium associated with human diarrhea, and chickens are considered to be one of the most important sources for human infection, with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce specific immune responses. The chitosan used for intranasal administration is a natural mucus absorption enhancer, which results in transgenic DNA expression in chicken nasopharynx.

[Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype avian influenza virus possessing H9N2 internal genes].

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008 (Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live bird market in East China during our epidemiological surveillance. To investigate the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with the characterization of the HPAIV.

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate.

Background: H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics.

Methods: In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2.

[Construction of recombinant fowlpox virus coexpressing HA gene from H5N1 avian influenza virus and chicken interleukin-2 gene and assessment of its protective efficacy].

The hemagglutinin (HA) gene from H5N1 avian influenza virus and the chicken interleukin 2 (chiIL-2) gene were inserted into a expressing vector p12LS to construct a recombinant transferring vector p12LSH5AIL2, in which HA gene under the control of the promoter Ps was in inverse tandem connection with the chiIL-2 gene under the control of the promoter PE/L. The p12LSH5AIL2 was then used to transfect the chicken embryo fibroblasts (CEF) pre-infected with a wild-type fowlpox virus 282E4 strain, to generate a recombinant fowlpox virus coexpressing the inserted HA and chiIL2 genes (rFPV-H5AIL2). The rFPV-H5AIL2 was obtained and purified by blue plaque screening on the CEF.

[Phylogenetic analysis of the neuraminidase genes of subtype N1 viruses in domestic ducks in eastern China].

To examine the phylogenetic information regarding the gene pool of AIV in domestic ducks in eastern China, the NA genes of twenty-six viruses isolated during 2002-2006, including two H1N1 strains, tenH3N1 strains and fourteen HSN1 strains, which reflected the predominant N1 subtype viruses were subjected to phylogenetic analysis. The results indicated that AIVs of N1 subtype circulating in domestic ducks in eastern China were undergoing a gradual evolution. Analysis of the deduced amino acid sequences revealed that NAs from all isolated H5N1 viruses had a 20-aa deletion in the stalk region (residues 49-68), whereas no deletion was seen in the NAs from other HA subtype viruses.

[Biological characteristics and sequence analysis of fusion genes of Newcastle disease virus isolates].

Twenty Newcastle disease virus (NDV) strains were isolated from chickens and geese in the field outbreaks during 2005 and 2006 in some regions of Jiangsu and Guangxi province. Assessment of the virulence by MDT and ICPI, RT-PCR and sequence analysis of fusion protein gene were used to compare the properties of NDV isolates. The results indicated that MDT and ICPI of the isolates were 45.

[Role of amino acid residues at positions 322 and 329 of hemagglutinin in virulence of H5N1 avian influenza virus].

Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys).

A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses.

To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank.

[Construction and immunogenicity of attenuated Salmonella typhimurium stably harbouring DNA vaccine against Newcastle disease virus].

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing.

Virulence of H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene.

More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene.

Comparison of virulence-associated traits between a UPEC strain HEC4 and a APEC strain E058.

Since avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar challenges when establishing infection in the extra-intestinal locations of the hosts, they may share a similar content of virulence genes and capacity to cause disease. One APEC and one UPEC isolates were compared by their content of virulence genes and other traits. The two strains showed overlap in terms of their virulence genotypes, including their possession of certain genes associated with a large transmissible plasmid of APEC, and also shared some biochemical activities.

[Development and characterization of monoclonal antibodies against H7 hemagglutinin of avian influenza virus].

Aim: To prepare monoclonal antibodies (mAbs) against the hemagglutinin protein of H7 subtype of avian influenza virus (AIV).

Methods: (6-8 weeks old) BALB/c mice of were immunized endermicly with H7 subtype of AIV. The splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells after the last immunization.

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses.

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine.

[Identification of APEC genes expressed in vivo by selective capture of transcribed sequences].

Direct screening of bacterial genes expressed during infection in the host is limited, because isolation of bacterial transcripts from host tissues necessitates separation from the abundance of host RNA. Selective capture of transcribed sequences (SCOTS) allows the selective capture of bacterial cDNA derived from infected tissues using hybridization to biotinylated bacterial genomic DNA. Avian pathogenic E.

[Attenuation of a genotype VIId Newcastle disease virus ZJI strain of goose origin by reverse genetics].

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain isolated from an outbreak in the goose, seven pairs of primers were designed to amplify cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of NDV ZJI strain. The pNDV/ZJI with three helper plasmids, pCI-NP, pCI-P and pCI-L, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, infectious NDV ZJI strain was successfully rescued.

[Analysis of in vitro delivering CD8+ T epitopes by attenuated bacteria].

Aim: To analyze the efficiency of delivery for CD8(+) T cell epitopes by recombinant bacteria vectors.

Methods: The recombinant E. coli strain 13A or 25A and recombinant Salmonella typhimurium strain SL7207 expressing CD8(+) T cell epitopes of Lymphocytic Choriomeningitis Virus (LCMV) and Ovalbumin (OVA), which were fused with green fluorescent protein (GFP) marker at the C-terminal, were infected into LK(b) cells, LL(d) cells or bone marrow dendritic cells (BMDC).

[Biological significance of amino acids deletion in NA stalk of H5N1 avian influenza virus].

It has been reported that NA gene of some H1N1 Influenza A virus strains isolated since 1933 is characterized by a deletion of 11 to 16 amino acids in the stalk. The spontaneous mutant in NA stalk of H1N1 virus lacks enzyme activity with large substrate (fetuin) but not with small substrate (sialyllactose). Recently, H5N1 virus also has been found that NA has the same unique mutation in the stalk, a deletion of 15 to 20 amino acids.

[Preparation and characterization of monoclonal antibodies against chicken interferon-gamma].

Aim: To prepare monoclonal antibodies (mAb) against chicken interferon-gamma (ChIFN-gamma).

Methods: By using lymphocyte hybridoma technique, the inclusion body of the recombined bacteria, BL21(DE3) (pET-ChIFN-gamma), was harvested and used to immunize BALB/c mice. With the purified GST-ChIFN-gamma as detecting antigen, mAbs against ChIFN-gamma were prepared, and positive hybridoma clones were screened by indirect ELISA.

[The deletion of nucleotides of NS gene from 263 to 277 of H5N1 increases viral virulence in chicken].

Since 2000, more and more NS fragment of H5N1 subtype influenza A virus had a unique deletion of nuleotides from 263 to 277. In order to investigate the biological significance of the mutation, four recombinants, RWSN-848, RWSN-m848, RWSN-248 and RWSN-m248, were generated via eight-plasmid reverse genetic system. These reassortants had the same inner gene and outer gene derived from A/WSN/33(H1N1) and A/D/SD/04(H5N1), respectively.