构建了ORF2蛋白原核表达体系Rosetta-pMAL-c5X-ORF2,经IPTG诱导表达后,对重组蛋白进行SDS-PAGE和Western blot分析,并采用镍柱亲和层析法进行纯化,以该蛋白为抗原建立液相蛋白芯片检测方法.结果显示:本试验成功表达了可溶性ORF2蛋白,具有良好的抗原性.液相蛋白芯片检测方法对猪常见的其他疾病阳性血清无交叉反应,其批内、批间变异系数分别为5％和6.6％.对102份临床血清样本检测结果显示,该方法与商品化ELISA试剂盒符合率为93.1％,关联性卡方检验显示2个方法具有一致性(P＜0.01).本试验为临床HEV血清抗体的检测提供了一种特异、灵敏的新型快速检测技术,为建立猪病多重检测方法提供了基础.

To investigate the infection situation and prevailing characteristics of swine hepatitis E (HE) in Guangdong,a total of 73 bile samples from pigs in Guangdong province were tested for the partial ORF2 gene of HEV by RT-nPCR.The positive of HEV RNA was 6.8％(5/73) and a high similarity (87.2％-99.4％) of partial ORF2 sequence was observed among 5 samples.The viral sequences belonged to genotype 4,subtypes 4b and 4 h.One complete genome of a subtype 4b HEV isolate GZHD was sequenced.Comparative sequence analysis showed that GZHD was closely identical to swGX40(EU676172),sharing 96.5％ nucleotide sequence identity and 93.9％ and 94.8％ nucleotide sequence identity with SS19 (JX855794),CV~Sie10 (LC042232).Above all,it revealed that the current epidemic HEV strain is still the genotype 4 in Guangdong,and offers molecular evidence for uncovering the interspecies transmission of HEV.

In order to establish a high-throughput,reproducibleand highly specific method to detect swine reproductive and respiratory syndrome virus(PRRSV) antibodies,the purified non-structural protein 7 (Nsp7) recombinant protein was used as an antigen to establish a fluorescent microsphere-based immunoassay(FMIA).SDS-PAGEand Western-blot analysis showed that PRRSV Nsp7 recombinant was soluble and had good antigenicity.The intra-assay and inter-assay coefficient of variation ofMFIA was 3.2％ and 4.2％,respectively,and there were no cross-reactions with the positive sera of other swine diseases.When the concentration of mAb was as low as 1 ng/mL,the method still had high sensitivity.These results indicate that the FMIA is rapid,sensitive and specific for detecting antibodies against swine PRRSV.

Nonstructural protein 7 (nsp7) of porcine reproductive and respiratory syndrome virus (PRRSV) is considered to be a suitable reagent for the development of serological diagnostic assays. It can be expressed as a soluble recombinant protein in Escherichia coli, and its antibody response may continue up to 202 days post-infection. Furthermore, the region encoded by nsp7 is highly homologous among various strains within the genotype, and the results of nsp7-based enzyme-linked immunosorbent assay (ELISA) showed high agreement with previous Idexx ELISA results. All these evidences suggest the existence of important epitopes on nsp7, though the characteristics of these epitopes remain unclear. In the present study, we prepared three monoclonal antibodies against nsp7 protein and used them to screen the epitope-distribution characteristics of PRRSV nsp7 protein by phage-display technology. We identified a linear epitope NAWGDEDRLN at amino acids 153–162 type II PRRSV nsp7β subunit. This newly defined epitope showed excellent reactivity with PRSSV-positive serum samples. These results further our understanding of the antigenic structure of nsp7 protein, and provide efficient reagents for PRRSV serological tests.

To express the HA and HA1 proteins of H3N2 swine influenza virus,the HA and HA1 genes were amplified by RT-PCR and cloned into prokaryotic expression vector pMAL-c5X.Recombinant plasmid pMAL-c5X-HA and pMAL-c5X-HA1 were transformed to E.coli Rosetta (DE3)and induced with IPTG. The HA and HA1 proteins were expressed successfully in the forms of soluble proteins and inclusion bodies.What′s more,both of the HA and HA1 proteins could be recognized by HA antibody of H3N2 sub-type,it revealed that the HA and HA1 proteins have good antigenicity.

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.

The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region.

为了解广东地区猪流感的流行和变异情况,本研究于2013年11月～12月从广东省5个猪场中采集疑似流感症状的猪鼻拭子115份,接种10日龄SPF鸡胚,分离得到一株猪流感病毒(SIV),对其进行血凝和血凝抑制试验、RT-PCR检测及全基因测序分析,结果表明该分离株为H1N1亚型,将其命名为A/Swine/Guangdong/L2/2013(H1N1).HA蛋白裂解位点序列为PSIQSR ↓ GL,具有典型的低致病性流感病毒特征.系统遗传进化分析显示,该病毒与A/swine/Shanghai/2/2005 (H 1N1)亲缘关系密切.小鼠的致病性试验表明该病毒株可以直接感染小鼠,导致小鼠轻微临床症状和肺脏组织病理学变化,但未在其他组织器官中分离到病毒,并且对小鼠无致死性,为低致病性SIV.该病毒的分离鉴定为广东地区SIV的流行特点和变异情况提供实验依据.

Hepatitis E virus (HEV) infection is widespread in China, but few studies have been carried out in Guangdong Province. This study aimed to characterize the prevalence of HEV infections among swine, swine farmers and the general population in Guangdong Province. We conducted an epidemiological study that included swine, swine farmers and health examination attendees in Guangdong from 2011 to 2013.. The overall seroprevalence of anti-HEV antibodies in swine was 64.7%. The results revealed that growing pigs, sows and boars (OR ranges from 3.5 to 21.5) have a higher risk than nursery pigs. HEV RNA in swine bile showed that HEV is epidemic in swine in the Pearl River Delta, with the highest prevalence of 22.73% in Foshan. Some genomes of HEV strains from each district were sequenced. Phylogenetic analysis of partial open reading frame 2 (ORF2) shows that they belong to genotype IV and are most closely related to isolates from China. In total, 307 participants were enrolled in the study, including 114 swine farmers and 193 attendees from hospitals. IgG anti-HEV was detected in 48.25% of swine farmers and in 38.34% of the general population. Seroprevalence rates were almost stratified by age, with a higher positive rate for males compared to females across all age groups. Women on swine farms appeared to have a lower risk of infection compared to the general population, revealing that the risk factors for HEV infection are not unique. The results suggested that there were other risk factors for HEV infection. HEV infection is prevalent in Guangdong, but due to the small sample sizes, more investigations are needed to assess the potential impact of HEV infection, and many additional risk factors should be considered.

Infection by Hepatitis E virus (HEV), as a zoonotic disease virus, is well studied in pigs in China, but few studies in pets have been performed. This study was designed to characterize the prevalence of HEV infection among pet dogs and cats in major metropolitan areas of China. We conducted a seroepidemiological survey from 2012 to 2013 in 5 developed cities, Beijing, Shanghai, Canton, Shenzhen and Macao, by enzyme-linked immunosorbent assay (ELISA). The overall HEV seroprevalence in 658 dog and 191 cat serum samples was 21.12% and 6.28%, respectively. The analysis in dogs suggested that there were significant differences among cities, and the positive rate of HEV-specific antibody in all cities ranged from 6.06% (Shenzhen) to 29.34% (Beijing). Older pet cats have a high risk (OR, 10.25) for HEV seropositivity, but no strong relationship was observed between different genders and age groups. Additionally, it was revealed that stray dogs, omnivorous pet dogs and pet cats who share food, such as kitchen residue, with the general population would have a higher risk for HEV seropositivity. The odds ratios for these groups are 2.40, 2.83 and 5.39, respectively, compared with pet dogs and cats fed on commercial food. In this study, we first report that HEV is prevalent in pet dogs and cats in several large cities in China. Swill and kitchen residue may be a potential risk for HEV transmission from human to pets. As the sample size was relatively small in this study and may not be fully representative of China, further investigation is required to confirm the conclusions.

为了研究猪源戊型肝炎病毒（swHEV）ORF3部分基因的抗原性，尝试通过G418的筛选，建立出稳定表达ORF3蛋白的真核细胞系。试验通过设计引物，扩增 swHEV ORF3部分基因片段，长度360bp。使用pMD18-T载体克隆，双酶切后插入pcDNA3.1真核表达载体。质粒经双酶切鉴定阳性后，转染真核细胞系HepG2，并进行G418筛选。筛选出HepG2-ORF3真核表达细胞系后，通过间接免疫荧光检测其表达情况，同时进行HepG2-ORF3细胞系的形态学和生长曲线的观察。实验最终证实成功建立HepG2-ORF3真核表达细胞系。

戊型肝炎(Hepatitis E) 是由戊型肝炎病毒(Hepatitis E virus，HEV) 引起的以黄疸为主的急性病毒性肝炎。目前研究表明, 戊型肝炎是一种人兽共患病。ORF2是戊型肝炎的第二开放阅读框，主要编码核衣壳蛋白，具有抗原性和免疫原性，是研究戊型肝炎病毒致病机理和疫苗应用方面的重点对象。本综述旨在综合近年来对ORF2的相关研究进展，为后续ORF2的研究提供一定的引导和参考作用。