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. NS1 protein is the key protein of the influenza virus against host innate immunity. The interaction between viruses and organisms is a complex and dynamic process, in which they restrict each other, but retain their own advantages. In this review, we start by introducing the structure and biological characteristics of NS1, and then investigate the factors that affect pathogenicity of influenza which determined by NS1. In order to uncover the importance of NS1, we analyze the interaction of NS1 protein with interferon system in innate immunity and the molecular mechanism of host antagonism to NS1 protein, highlight the unique biological function of NS1 protein in cell cycle.

[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. The EID50 and TCID50 tested in SPF chick embryo and MDCK cells of a mutant rSdelta158 with deletion of glycosylation site at position 158 were slight lower than that of wild type rescued virus rS, and the plaque diameter of rSdelta158 was significant smaller than that of rS. The EID50 and TCID50 of mutants rSdelta169 and rSdelta290 with deletion of glycosylation sites at position 169 and 290, respectively, were slight higher than that of wild type rescued virus rS, the plaque diameters of rSdelta169 and rSdelta290 were similar as that of rS, but the plaque numbers of rSdelta169 and rSdelta290 were 10-fold higher than that to rS. On the other hand, the rSdelta158, rSdelta169 and rSdelta290 showed similar growth rate in chicken embryo fibroblast as rS. All viruses remained high pathogenicity to SPF chickens. Therefore, the growth of AIV can be affected by changes of glycosylation sites in HA protein, by which the effect is variable in different cells.

[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. All isolates had a S to N substitution at residue 31 in the M2 gene, which is related to the resistance phenotype of adamantanes. The key molecular features of 16 AIV isolates from different hosts were same. Genome phylogenetic analysis revealed that all 16 H9N2 subtype AIVs originated from F98-like virus as backbone and formed two new genotypes through reassortment with HA gene of Y280-like virus and PB2 and M genes of G1-like virus. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

[Construction and rescue of infectious cDNA clone of pigeon-origin Newcastle disease virus strain JS/07/04/Pi].

Based on the complete genome sequence of pigeon-origin Newcastle disease virus strain JS/07/04/ Pi(genotype VIb), nine overlapped fragments covering its full-length genome were amplified by RT-PCR. The fragments were connected sequentially and then inserted into the transcription vector TVT7/R resulting in the TVT/071204 which contained the full genome of strain JS/07/04/Pi. The TVT/071204 was co-transfected with three helper plasmids pCI-NP, pCI-P and pCI-L into the BSR cells, and the transfected cells and culture supernatant were inoculated into 9-day-old SPF embryonated eggs 60 h post-transfection. The HA and HI tests were conducted following the death of embryonated eggs. The results showed that the allantoic fluids obtained were HA positive and the HA could be inhibited by anti-NDV serum which indicated that the strain JS/07/04/Pi was rescued successfully. The rescued virus rNDV/071204 showed similar growth kinetics to its parental virus in CEF. The successful recovery of this strain would contribute to the understanding of the host-specificity of pigeon-origin NDV and to the development of the novel vaccines against the NDV infection in pigeons.

[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 (+). The purified rGST-CjaA protein was used as an immunogen in 8-week-old BALB/c mice, and injected subcutaneously. The purified rHis-CjaA protein used as a detecting antigen for screening mAbs against CjaA was prepared. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. RESULTS: The recombinant expression plasmids, pGEX-6p-1-cjaA and pET30a(+)-cjaA were obtained. The sizes of the recombinant proteins, rGST-CjaA and rHis-CjaA, were consistent with their predicted size. Specific reaction was found between CjaA positive serum and expressed protein by Western blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2B6, 3C2 and 4F11, secreting mAbs against CjaA were obtained. Their immunoglobulin subclasses were all IgG1. The ELISA titers of the ascites fluid were 1:1×10(5);, 1:2×10(5); and 1:4×10(5);, respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-CjaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with CjaA and not the tags for the expression vectors. CONCLUSION: The successful preparation of three mAbs specific for the CjaA protein lays the foundation for further study regarding the biological characteristics of CjaA and the pathogenesis of C. jejuni.

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. Chickens immunized with chitosan-DNA nanoparticles, which carried a gene for the major structural protein FlaA, produced significantly increased levels of serum anti-Campylobacter jejuni IgG and intestinal mucosal antibody (IgA), compared to those treated with chitosan-DNA (pCAGGS). Chitosan-pCAGGS-flaA intranasal immunization induced reductions of bacterial expellation by 2-3 log(10) and 2 log(10) in large intestine and cecum of chickens, respectively, when administered with the isolated C. jejuni strain. This study demonstrated that intranasal delivery of chitosan-DNA vaccine successfully induced effective immune response and might be a promising vaccine candidate against C. jejuni infection.

[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. According to the newest unified nomenclature system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M) displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08 might be a natural reassortant virus. The phylogenetic analysis further indicated that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern China might provide the internal genes for Dk/ SD/009/08.

[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 45.3h - 58.2h and 1.61 - 2.00 respectively, which confirmed that the all NDV isolates were highly virulent. And their hemagglutinin were not resistant to heat and belonged to fast pattern of elution. The results of nucleotide sequencing and phylogentic analysis of fusion protein gene showed that the twenty strains shared homology from 79.7% to 100% among themselves, from 78.1% to 83.4% and from 80.2% to 90.1% with NDV LaSota, F48E8, respectively. The putative amino acid sequences of fusion protein at the cleavage sites of all the isolates were 112R-R-Q-R/K-R-F117, with the motif characteristics of the virulent NDV strain, which was in accordant with the results of assessment of the pathogenicity. The phylogentic tree based on sequences of fusion protein gene variable regions (47-420nt) revealed that the 18 strains belonged to sub-genotype VIId and the others belonged to an old genotype III of NDV, revealing that subgenotype VIId virus was responsible for the NDV outbreaks in some regions of Jiangsu and Guangxi promince recently.

[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. The viruses of H3N1 and H1N1 might have a propensity for reassortment of NA genes, whereas no direct evidence of reassortment of NA gene was obtained in H5N1 viruses.

[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. The in vitro expression of HA gene by rFPV-H5AIL2 was detected in the recombinant fowlpox virus-infected CEFs with an indirect immunofluorescence assay, and the expression of the chiIL2 gene by rFPV-H5AIL2 was confirmed by detection of the chiIL2 mRNA by RT-PCR and by detection of chiIL2 by the indirect immunofluorescence assay. Experiments on SPF and commercial chickens demonstrated that the titer for HI antibodies induced by the rFPV-H5AIL2 was significantly higher than that by the rFPV-HA. The group immunized with the rFPV-H5AIL2 exhibited the similar ratios of protective efficacy and virus shedding as the group immunized with the rFPV-HA in SPF chicken. However, in commercial chicken, the group immunized with the rFPV-H5AIL2 generated significantly higher protection against H5N1 avian influenza virus challenge and lower virus shedding than the group immunized with the rFPV-HA. This study paved the way for further development of a new AIV recombinant vaccine.

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. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.

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. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A). RESULTS: In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found. CONCLUSION: The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.

[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). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.

[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. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.

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. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263-277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID50 or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD50 in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.

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. Study of the pathogenicity of UPEC in chicks showed the similar symptoms and lesions compare to those caused by APEC. Based on these results, the potential whether APEC might serve as a reservoir of plasmid-linked and virulence genes for UPEC should be considered.

[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. Hybridoma cells were screened by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The reactivity and specificity of mAbs were evaluated by HI test and Western blot assay. RESULTS: Four hybridoma cell lines secreting specific mAbs named 2E2, 2A4, 5F5 and 7G5 were developed. The HI titers of these mAbs were 5 x 2(7)-5 x 2(11), and the immunoglobulin subclass of 2E2 was IgM, that of 2A4 was IgG1, and that of 5F5 and 7G5 was IgG2a. Western blot analysis confirmed that the mAbs only reacted with M(r) 75 000 HA protein of H7 subtype of AIV but did not react with the proteins of Newcastle disease virus (NDV). The results of HI reactivity assay suggested that 2E2, 5F5 and 7G5 only reacted with H7 subtype of AIV but did not react with other subtypes of AIV, NDV and infectious bronchitis virus (IBV). However 2A4 reacted not only with H7 subtype of AIV but also with H15N8 reference strain of AIV at low HI level. CONCLUSION: These mAbs can be used as a useful tool to analyze the HA structure of AIV. They also provide the effective reagents for the rapid detection of H7 subtype of AIV.

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. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.

[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. coli strain E037 (serogroup O78) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. Three-week-old white leghorn specific-pathogen-free chickens were inoculated into the right thoracic air sac with a 0.1 mL suspension containing 10(7) CFU of APEC strain E037. Total RNA was isolated from infected tissues (pericardium and air sacs) 6 or 24h postinfection and converted to cDNAs. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-pathogenic E. coli K-12 strain ) transcripts, pathogen-specific cDNAs were identified. Randomly chosen cDNA clones derived from transcripts in the air sacs or pericardium were selected and sequenced. The clones, termed aec, contained numerous APEC-specific sequences. Among the distinct 31 aec clones, pathogen-specific clones contained sequences homologous to known and novel putative bacterial virulence gene products involved in adherence, iron transport, lipopolysaccharide (LPS) synthesis, plasmid replication and conjugation, putative phage encoded products, and gene products of unknown function. Overall, the current study provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression of a pathogenic E. coli strain in a natural animal host during the infectious process.