Construction and biological characteristics of H5N1 avian influenza viruses with different patterns of the glycosylation sites in HA protein / 病毒学报

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 / 病毒学报

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 / 病毒学报

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.

Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype avian influenza virus possessing H9N2 internal genes / 病毒学报

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 / 病毒学报

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 / 病毒学报

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 / 病毒学报

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.

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Construction and immunogenicity of attenuated Salmonella typhimurium stably harbouring DNA vaccine against Newcastle disease virus / 病毒学报

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.

Role of amino acid residues at positions 322 and 329 of hemagglutinin in virulence of H5N1 avian influenza virus / 病毒学报

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.

Isolation and Identification of Porcine Circovirus Type 2 Virus from a Domesticated Wild Boar / 微生物学通报

The nucleic acid fragment of porcine circovirus type 2(PCV2)was amplified using PCR from the tissues of a domesticated wild boar with suspected postweaning multisystemic wasting syndrome(PMWS).The sample were then inoculated into the Dulac cells and passaged for 8 generation.The 4 nucleic acid fragments covered the complete genome for PCV2 were obtained by over-lapping PCR and sent to sequence.The sequence of genome was compared with other 9 strains originated from piglets in the same area.The result showed that these strains were in high homology.

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene / 中国病毒学

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,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,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

Combination of direct-ELISA and PCR for the rapid detection and identification of Salmonella spp / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a protocol for the rapid detection of Salmonellae.</p><p><b>METHODS</b>A mono-antibody-based direct-ELISA and PCR methods for the detection of Salmonella were developed previously. This study assessed the accuracy of both direct-ELISA and PCR methods for the rapid detection of Salmonella and set up a new detection protocol.</p><p><b>RESULTS</b>The sensitivity of the PCR method was higher than that of direct-ELISA method. In the 2002 spring physical examination for employees, 1 546 human fecal samples were examined by the combination of direct-ELISA and PCR method. Compared with the results of national standard method, the sensitivity and specificity of direct ELISA was 100% and 97.14%, respectively, while those of PCR method reached both 100%. It also indicated that combination use of two methods could give positive report within 40 hrs, and also achieve high sensitivity and specificity.</p><p><b>CONCLUSIONS</b>Based on the results obtained, a protocol for the rapid detection of Salmonella was developed. The first step is to us direct-ELISA method to screen the large number of samples, and then use PCR method to validate the ELISA positive samples, and the final step is, if needed, is to use the national standard method to determine the serotypes of Salmonellae.</p>

Construction of recombinant fowlpox virus expressing chicken IL-2 and assay of biologic activity of the product in vitro / 生物工程学报

In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.

The effects of estrogen-like products in milk on prostate and testes / 中华男科学杂志

<p><b>OBJECTIVES</b>To study the effects of estrogen-like products in milk on the male reproductive system.</p><p><b>METHODS</b>The incidence rates of testicular and prostatic cancers in 42 countries (regions), collected from the cancer incidence in five continent database (1988-1992), were correlated with the relative food consumption in these countries, which was provided by FAO database (1961-1990); two-generation reproduction test was taken by using Wistar rats according to FDA standard.</p><p><b>RESULTS</b>Among the food items examined, milk was most closely correlated (r = 0.711) with prostatic cancer incidence, followed by meat and coffee. As for testicular cancer, cheese was most closely correlated (r = 0.804) with the incidences in ages of 20-39, followed by animal fat and milk. No significant decreases of reproductive organ weight were found after the rats had been fed a lot of milk for long time.</p><p><b>CONCLUSIONS</b>The results suggest that further studies should be taken on the effects of estrogen-like products in milk on the male reproductive health.</p>

ClONING OF NP, P AND L GENE OF NEWCASTLE DISEASE VIRUS OF GOOSE ORIGIN AND IDENTIFICATION OF P GENE EXPRESSION / 微生物学通报

NP, P and L gene of Newcastle disease virus of goose origin were amplified and cloned into pGEM-T easy vector and then subcloned into pCI-neo expression vector respectively, the positive clones were identified by enzyme cutting, PCR and sequencing. GFP reporter gene was inserted into the downstream of recombinant expression plasmid of P gene, which of stop codon was deleted. The experiment of transfection of P and GFP recombinant plasmid on COS-1 cells and CEF showed that GFP gene expressed, and this demonstrated that P gene was also expressed. This research may be helpful for further study of reverse genetics and functional genome of NDV of goose origin.

Genetic Characterization and Antigenic Analysis of Hemagglutinin-neuraminidase Glycoprotein of Newcastle Disease Virus Isolates / 微生物学通报

Twenty Newcastle disease virus(NDV)strains were isolated from diseased chicken and geese in field outbreaks during 2005 and 2006 in some regions of Jiangsu and Guangxi,and the antigenic analysis of the all NDV isolates had been done based on the reaction spectrum with a panel of monoclonal antibodies to the HN glycoprotein.The entire ORFs encoding HN protein of these NDV isolates were amplified by RT-PCR successfully,cloned and sequenced.The resultant sequences of HN genes of 13 isolates of chicken origin and 7 isolates of goose origin were gained and analyzed.The results of reaction spectrum showed that there were some distinct differences in the antigenic epitopes among the 20 NDV isolates.And the sequences revealed that the coding regions of the HN genes of these isolates all consisted of 1716 nt characteristic of virulent strains of NDV,coding for 571 amino acids.Neucleotides sequence homology were found to be from 94.8%to 100%among 18 NDV isolates of genotypeⅦ,and the neucleotides sequence homology between all the isolates and the other genotypeⅦstrains of recent years in China ranged from 92.1%to 99.6%.The deduced amino acid sequences and the receptor-binding regions of HN proteins between the NDV isolates of chicken origin and of goose origin were compared and analyzed.The results showed that some unique amino acid substitutions were found in the genome of the NDV isolates,and the close genetic similarity provided evidence for epidemiological linkage between the NDV isolates of chicken origin and of goose origin in the same period.

Establishment of Reverse Genetics System for NDV Isolated from Goose / 微生物学通报

Eight fragments were amplified and cloned into pCR2.1 vector with the designed primers.The fragments,amplified with primer Ⅰ to Ⅶ,were subcloned into transcription vector to construct the plasmid pNDVZJI which contained the full-length cDNA of NDV ZJI strain.The eukaryotic expression vector pCI-L was constructed by subcloning the fragments,amplified with the primer Ⅴ,Ⅵ and Ⅷ,into the expression vector pCI-neo.The full-length cDNA clone,pNDVZJI,with three helper plasmids,pCI-NP、pCI-P and pCI-L,were cotranfected into BSR-T7/5 cell expressing T7 RNA polymerase.After inoculation of transfected cell culture into embryonated chicken eggs from specific pathogen free(SPF)flock,The NDV of ZJI strain was rescued successfully,which laid a good foundation for the further related research.

Effect of Fusion Protein Cleavage Site Mutations of NDV ZJ1 Strain on Its Fusion Activity / 微生物学通报

NDV strain ZJ1 strain , a highly virulent NDV strain, has been prevalent among the waterfowls in China mainland in the past years. Multi-basic amino acid sequence distribute in the protease cleavage site of F protein of this strain. Recombinant expressing plasmid pCI-FT, was generated by converting multi-basic amino acid sequence of 112, 115, 117 of the protease cleavage site of F_ 0 protein, to the non-basic amino acid sequence characteristic of avirulent NDV strain. The result from co-expression of mutant or parental F protein with homologous HN protein in COS-1 cells revealed that both mutant and parental F protein had fusion activity. The result from co-expression of mutant or parental F protein with homologous HN protein in CEF cells showed that the cleavage activity of mutant F protein was significantly reduced. The study built a foundation for mutagenesis of amino acid sequence of the protease cleavage site of F_ 0 protein at the full-length cDNA clone level, study on factors contributing to virulence and construction of candidate vaccine strain, and so on.

Deletion of Nucleotides of NS Gene from 263 to 277 Decreases the Viral Anti-IFN Ability of H5N1 / 微生物学通报

Since 2000,most of H5N1 subtype influenza A virus had a unique mutation of NS gene with 15base pair deletion from 263 to 277. In order to investigate the bio-characteristics of this mutation,two different NS recombinants,RWSN-248 and RWSN-m248,were generated via plasmid rescue from A/WSN/33(H1N1) and A/SD/04(H5N1). RWSN-248 had a higher viral titer than RWSN-m248 in MDCK and COS-1 cells that have an IFN response,but they had the similar growth ability in Vero cells that lack an IFN response. Both of two recombinants grew well in embryonated chicken eggs and had the similar viral titer and MDT. The results above revealed that the deletion from 263 to 277 sites of NS gene did not influence viral virulence to but decreased viral anti-IFN ability of H5N1.