Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive and respiratory diseases in sow herds and piglets. The emergence of ORF5 RFLP 1-7-4-like (NADC34-like) PRRSV strain in China has brought a new round of challenges to PRRSV prevention. Methods: In addition, recombinant adenovirus vaccine candidates against the newly emerged NADC34-like strain were constructed in the study; the immunogenicity of the vaccine was investigated in piglets. After inoculation with PRRSV recombinant adenovirus, specific antibodies, neutralizing antibodies, and levels of IFN-gamma and IL-4 cytokines were detected in serum. Results: Thirty-five days after immunization, the levels of IFN-gamma and IL-4 cytokines in the pac-Ad5-34-GP3, pac-Ad5-34-GP5, and pac-Ad5-34-GP35 experimental groups were significantly higher (p < 0.05) than those of the PBS and the adenovirus group. All vaccines can cause corresponding Th1 and Th2 immune responses based on animal experimental results. After the challenge, no obvious clinical symptoms were observed in the immune groups compared with the control group, vaccinated animals could reduce the occurrence of viremia, and the occurrence of viremia was alleviated, with no obvious pathological changes in the lungs, indicating that recombinant adenovirus vaccine could provide a good protective immunity and produce a good humoral and cellular immune response at the same time. Discussion: It shows that the recombinant adenovirus vaccine group has better protection against the virus. Provide vaccine reserve and theoretical support for the emergence of new PRRSV subtypes in China.

To explore the pathogenesis and transmission mechanism of influenza virus PR8F in the BALB/c mice so as to serve the development of new vaccines,the 8 plasmids (A/PR/8/34,stored in our lab) were point specifically mutated according to the eight-plasmid mutation system of influenza virus founded by Paulina.A mutation PR8F of H1N1 influenza virus were rescued by reverse genetics.Mice were inoculated with mutation PR8F of influenza virus at dose of 106 TCID50/100 μL by the nasal cavity.The clinical manifestation and body weight changes were monitored.All the mice died at 4 7 days post inoculation.The main organs were obtained and pathological sections were observed.Viral RNA was extracted and then detected by qPCR for the virus residue of mutation PR8F in the organs and its amplification efficiency in lung.The result indicated that the mutation PR8F had a 100％ mortality rate in mice;there were significant differences in the amount of viral residues in different mice organs.The virus retained the most in lung and copied in exponential form.The mouse infection model of influenza virus (H1N1) mutation has been successfully established which can lay a foundation for the study of the pathogenesis and transmission mechanism of H1N1 influenza virus.

In order to rapidly detect Torque tenocanis virus (TTCV),the SYBR Green I real-time PCR method was proposed to detect TTCV with a pair of specific primers were synthesized according to the TTCV ORF7 gene,and the reaction conditions and reaction system were optimized.The results showed that the Ct value of the proposed assay linearly related to the standard template in the range of 5.82×109 copies/μL to 5.82×103 copies/μL.This assay was only positive for TTCV detection,while showed no specific amplification for RABV,CCV,CPV,CDV,RV;the sensitivity of the assay was 5.82×103 copies/μL.The detection for 27 canine serum samples showed that the positive rate of the proposed real-time PCR method was 11.11％.While,the positive rate of conventional PCR was 3.7％.In this study,the developed TTCV SYBR Green I real-time PCR could provide ahigh-throughput quantitative detection for the research of diagnosis and pathogenesis of TTCV.

The Torque teno canis virus (TTCaV) is a small virus with circular single-stranded DNA that has been reported to cause infections in dogs. The present study aimed to identify the presence of TTCaV in blood samples obtained from domestic dogs, and examine its diversity and evolution of the genomes. Five strains of TTCaV were detected, and the overall prevalence was found to be 7% (28/400). Phylogenetic analysis showed that the five genomes were closely clustered with the previously known Cf-TIV10 and LDL strains and formed a Thetatorque virus. Homology analysis of the whole genome showed a sequence identity of 94.6%-96.8% among the five genomes. The percent sequence similarity among the five complete genomes ranged from 95.3% to 97.4% and from 95.1% to 97% compared to the Cf-TTV10 and LDL strains respectively. The ORF1-encoded amino acid sequences showed 94.4%-97.2% identity among the five isolates. Our findings suggest that the TTCaV has a large genetic diversity and showed that TTCaV and canine parvovirus (CPV) co-infection exists in China. Further studies on the pathogenicity of TrCaV are required.

A SYBR Green Ⅰ real-time PCR method for porcine parvovirus 7 (PPV7) was established using primers derived from the published sequences.A plasmid containing PPV7 capsid gene was constructed and served as a standard template to generate the standard curve.The standard curve produced a good linear relationship between Ct value and concentrations of the standard template at a range of 5.00 ×109 copies/μL to 5.00 × 103 copies/μL.The specificity assay showed no amplification of PRRSV,PCV2,JEV,PRV and FMDV except PPV7.The sensitivity of this method was 5.00×101 copies/μL.The reproducibility was high,and the variation coefficients of intra-assay and inter-assay were less than 1％.The positive rate was 71.88％ of samples collected from Guangxi province decected by this method,which was 4.55％ higher than the conventional PCR assay.This study could provide a new rapid quantitative method to detect PPV7.

Objective To investigate the molecular epidemiology of the Nsp2 and ORF5 genes of the porcine reproductive and respiratory syndrome virus (PRRSV) found in Guangxi.Methods In total,45 samples of PRRSV were collected from Guangxi in 2015,and the Nsp2 and ORF5 genes of 7 strains were used to determine their molecular epidemiology.Results The 7 samples tested positive for PRRSV according to RT-PCR.The 7 strains were 83.5％-88.7％ similar to VR-2332 and 62.1％-64.8 ％ similar to LV.The phylogenetic tree indicated that the 7 strains could be divided into 2 subgroups.Three strains were clustered in subgroup IV while the other 4 strains were clustered in subgroup VI.Sequencing of Nsp2 indicated that 6 strains had deletion of amino acid 29,which was similar to highly pathogenic PRRSV-1.The Nsp2 gene of GXBB11-2015 was found to have deletion of 20 amino acids and deletion of 150 bases.Conclusion Highly pathogenic PRRSV has become the predominant strain in Guangxi.Detection of deleted bases in Nsp2 provided new evidence of PRRSV mutations,laying a foundation for further study of the effect of gene deletion on pathogenicity.

目的 对2013年从广西分离的H9N2亚型禽流感病毒进行全基因测序,并分析其基因组和遗传进化特性,为进一步研究H9N2禽流感病毒的致病机制提供科学依据. 方法 运用RT-PCR方法克隆广西分离株H9N2亚型禽流感病毒8个基因片段,进行全基因测序,对测序结果进行序列比对和遗传进化分析. 结果 序列比对分析表明,H9N2亚型分离株的HA序列裂解位点为RSSR↓ GLF,受体结合位点为YWTNKLY且发生Q226G和G228R双重位点突变,NA耐药相关位点发生3处突变(E119V,R292W和R294G),M2蛋白跨膜区存在S31N变异;编码内部蛋白的6个基因与同年H7N9亚型分离株相似度为90.9％～97.6％.其分子遗传进化关系属于欧亚种系,其中HA基因属于Y280亚系,M基因属于G1亚系,PB2基因属于Korea亚系,PB1、PA、NA、NP和NS属于SH/F/98亚系. 结论 2013年广西分离株是由4种不同H9N2亚型禽流感病毒亚系发生自然重排的产物,属于低致病性禽流感病毒,但对抗流感药物存在一定耐药性,故应加强对禽农场中H9N2病毒流行株变异趋势和传播风险的监测.

The aim of this study was to explore the influence of mutation of different non-structural (NS) 1 amino-acid residues on the pathogenicity of influenza viruses and the function of NS1 virulence-related sites on the pathogenesis of influenza viruses. We analyzed segments of the NS1 protein gene and key sites related to virulence of influenza viruses based on a literature review. Fragments of the NS1 gene were cloned from the HIN1 subtype PR8F (non-mutated) and preserved by our research team with encoding sequence site-specific mutagenesis at aa42, aa8l, and aa149. Via a reverse genetics system, we rescued the mutant strains PR8F-42, PR8F-81, and PR8F-149, which were inoculated into chick embryos and could replicate stably after five passages. Efficiency of viral replication was measured by testing hemagglutination titers. BALB/c mice were inoculated With mutated or non-mutated PR8F (10(6) TCIDO(50)/100 μl for each mouse), respectively. The typical clinical manifestations (weight change and survival) were recorded. Autopsies, as well as observations of the pathologic features and pulmonary-tissue slices of mice that died after inoculation, were done. RNA of mouse lungs was extracted, and the residual quantity of virus in lungs was detected by quantitative polymerase chain reaction (qPCR). Results showed that mutation of NS1 at aa42 from Ser to Pro did not change the pathogenicity of PR8F in mice, but a low pathogenicity of PR8F occurred after mutation of NS1 at aa8l and aa149. The present study lays the foundation for further investigations of the function of NS1 pathogenicity-related sites in the pathogenesis of influenza viruses.

Objective To synthesize initial fragments of genes of the Newcastle disease virus (JL02/2000) in order to create a construct containing the full-length genome of the virus.Methods Initial fragments of the ND1 gene (226-4 916 bp) were divided into four smaller fragments designated ND1-1,ND1-2,ND1-3,and ND1-4,and each fragment was amplified with PCR.SOE PCR was used to splice ND1-1 and ND1-2 to an intermediate ND1-1-2.ND1-3 and ND1-4 were spliced to ND1-3-4.Two intermediates,ND1-1,2 and ND1-3,4,were assembled with SOE PCR to synthesize initial fragments of the ND1 gene,and the ND1 fragment was ligated to a pCI vector to construct the plasmid pCI-ND1.The target fragment was identified with sequencing as well as restriction enzyme digestion.Results ND1-1,ND1-2,ND1-3,and ND1-4 were amplified with PCR,yielding a product of about 1 200 bp.SOE PCR was used to synthesize two intermediates,ND1-1-2 and ND1-3-4,yielding a fragment of 2 200 bp each.An ND1 fragment of about 4 900 bp was synthesized using a second round of SOE PCR.The pCI-ND1 plasmid was identified with sequencing and restriction enzyme digestion,and the results of enzyme digestion and genetic sequencing were consistent with expectations.Conclusion Initial fragments of a gene were successfully spliced using SOE PCR.This work has laid the foundation for creating a construct containing the full-length genome of the Newcastle disease virus.

目的 为了探索经济、高效的腺病毒的纯化工艺,采用阴离子交换层析和分子筛层析两步法对H3亚型流感重组腺病毒pacAd-H3-EHA-H1HA1 new进行纯化,并评价纯化效果. 方法 利用7L生物反应器培养HEK-293细胞,用于对H3型流感重组腺病毒进行扩增;利用冻融的方法使病毒从细胞中释放,通过阴离子交换层析和分子筛层析法纯化病毒,用分光光度计检测纯化样品的纯度,并测定病毒的滴度和回收率. 结果 纯化后的样品经PCR鉴定正确,纯化前后的病毒滴度分别为5.6×109 TCID50/ml和1.1×1010 TCID50/ml,纯化后样品纯度(A260/A280值)分别为2.01和1.26,阴离子交换层析纯化的回收率为31.5％,分子筛层析纯化的回收率为81.4％,总回收率为25.6％. 结论 利用阴离子交换层析和分子筛层析两步法纯化H3亚型流感重组腺病毒的回收率和滴度高,该方法可用于重组腺病毒的纯化.

为建立可同时检测新疆出血热病毒(CCHFV)、裂谷热病毒(RVFV)、拉沙病毒(LSV)的荧光基因芯片,分剐针对CCHFV和RVFV的S基因、LSV的NP基因,应用生物学软件设计了3对引物与6条探针.运用多重PCR扩增和标记目的片段,将其与基因芯片杂交,最后扫描分析结果.分别对杂交温度与杂交时间进行条件优化,最后对基因芯片的特异性、敏感性和重复性进行评估.结果显示,设计的所有探针都可以与目的片段特异性杂交,探针1a与1b可检测到CCHFV,探针2a与2b可检测到RVFV,探针3a与3b可检测到LSV.相比多重PCR,荧光基因芯片检测CCHFV的灵敏度提高了10倍,检测RVFV的灵敏度提高了100倍,检测LSV的灵敏度提高了100倍.结果表明,本试验建立的同时检测3种出血热病毒的荧光基因芯片检测方法特异性高、敏感性强,可用于这3种病毒的快速检测与甄别.

To construct American serotype porcine reproductive and respiratory syndrome virus(PRRSV)vaccine containing glycosylated protein GP5 antigenic epitope,hepatitis B virus core protein(HBc)particle was used as a vector.The codons of HBc sequence were optimized and cloned into the pET-28a(+)to gain plasmid pEO-NEW.The GP5 gene was inserted into the major immunodominant region of HBc to gain recombinant expression plasmid pEO-LEORF5.It was transformed into Escherichia coli BL21(DE3)for expression under induction of IPTG at 22 ℃.SDS-PAGE,Western-blot and the transmission electron microscope were utilized to analyze the character and structure of the recombinant protein.After ultrasonic broken,fusion protein was purified by sucrose gradient centrifugation.The results showed that the recombinant expression plasmid pEO-LEORF5 was constructed correctly.The expressed fusion protein,with a molecular mass of about 32 ku,existed in both soluble and inclusion body forms.After purification,the fusion protein reached a purity of 67% and could be assembled automatically into virus-like particles(VLPs).The constructed HBc-GP5 VLPs candidate vaccine provided a new way of developing the PRRS vaccine.