低温电镜术证实了兔出血症病毒亚基因组颗粒的存在

从病兔肝细胞分离纯化的兔出血症病毒 ( RHDV)的低温电镜术显示出存在两种核心密度有明显差异的颗粒——高密度颗粒和低密度颗粒 ;而病毒的负染电镜术则显示出绝大多数病毒颗粒具有完整病毒颗粒的形态。结构分析表明高密度颗粒和低密度颗粒具有相同的衣壳结构 ,且不存在释放核酸的通道。对低密度颗粒核心区的密度分析表明相当数量的颗粒并非空衣壳。我们认为这部分低密度颗粒是含病毒亚基因组 RNA的病毒颗粒 ,从而为兔出血症病毒在复制过程中其 7.5kb基因组和 2 .2 kb亚基因组 RNA分别包装成病毒颗粒提供了结构证据。

慢性丙型肝炎病毒亚基因型载量检测分析研究

探讨慢性丙型肝炎(CHC)患者1b亚型及2a亚型RNA病毒载量水平与丙氨酸氨基转移酶(ALT)的相关性。选取2018年1月-2020年3月我院收治的125例慢性丙型肝炎感染患者作为慢性丙型肝炎感染组和健康体检正常者60例作为对照组。采用荧光定量基因探针技检测将C病毒分型将1b亚型与2a亚型。慢性丙型肝炎患者根据HCV-RNA的检测结果,分为丙型肝炎感染患者低病毒载量组(n=33,HCV-RNA<1.0×103 IU/mL)、丙型肝炎感染患者中病毒载量组(n=55,1.0×103 IU/mL≤HCV-RNA≤1.0×106 IU/mL)和丙型肝炎感染患者高病毒载量组(n=37,HCV-RNA>1.0×106 IU/mL);根据ALT的检测结果,ALT正常组(n=25,ALT<40 U/L)、ALT轻度升高组(n=48,40 U/L≤ALT≤80 U/L)和ALT明显升高组(n=52,ALT>80 U/L)慢性丙型肝炎组和对照组性别、年龄、体质指数及慢性丙型肝炎家族史等一般资料比较,。采用Pearson相关分析慢性丙型肝炎患者血清1b亚型及2a亚型表达水平与ALT,HCV-RNA的相关性。结果:高病毒载量组1b亚型(6.92±2.14 vs 5.53±1.90,4.05±1.20)及2a亚型(4.62±1.51 vs 3.25±1.27,1.96±0.81)表达水平明显高于中病毒载量组和低病毒载量组(P<0.01),且中病毒载量组1b亚型(5.53±1.90 vs 4.05±1.20)及2a亚型(3.25±1.27 vs 1.96±0.81)表达水平明显高于低病毒载量组(P<0.01)。相关分析显示,慢性丙型肝炎组和对照组性别、年龄、体质指数表达水平与ALT、HCV-RNA均呈正相关(r=0.858、r=0.471、r=0.524、r=0.502,P<0.001)。结论 慢性丙型肝炎患者血清1b亚型及2a亚型表达水平明显升高,与ALT、HCV-RNA均呈正相关,提示慢性丙型肝炎病情发生发展与1b亚型及2a亚型不同而不同。

J亚群禽白血病Hrb-1分离株env基因克隆及gp85杆状病毒表达载体的构建

自哈尔滨某送检患病鸡群中分离出一株病毒,经RT＿PCR检测、SPF鸡胚成纤维细胞增殖后,获取其前病毒DNA,采用依据原型毒株HPRS＿103cDNA序列设计并合成的一对引物,PCR扩增病毒的囊膜基因,连接pMD18＿T载体并转化大肠杆菌JM109,培养后提取质粒分别用HindIII,BamHI进行单酶切和双酶切鉴定,得到了阳性重组质粒pMD18＿T＿Hrb＿1/env,对其进行PstI酶切,回收包含J亚群禽白血病病毒株Hrb＿1gp85基因的997bp片段,应用BactoBac杆状病毒表达系统,将外源片段与线性化的杆状病毒载体pFastBacHTA进行连接,获得重组载体pFASTBacHTA/gp85,将该重组载体转化DH10Bac感受态细菌,在体内进行重组,经抗性和蓝白斑筛选,获得了杆状病毒重组载体Bacmid/gp85,为表达gp85并建立适于国内应用的ELISA诊断方法奠定了基础。

2种佐剂在猪圆环病毒2型基因工程亚单位疫苗中的效果比较

为给猪圆环病毒2型基因工程亚单位疫苗选择一种理想的佐剂,以增强机体对抗原的适应性免疫应答,协同提高动物免疫力。实验用水包油Merckinade SDA 25佐剂和双向Montanide ISA 206佐剂,分别按各自说明制备猪圆环病毒2型基因工程亚单位疫苗,再对两种佐剂的疫苗进行稳定性、黏度、安全及效力检验,比较评价出猪圆环病毒2型基因工程亚单位疫苗的理想佐剂。结果表明,两种佐剂疫苗的稳定性、黏度、安全均合格,无明显差异;但Montanide ISA 206佐剂疫苗较Merckinade SDA 25佐剂疫苗诱导抗体效价更高。据此,笔者认为,Montanide ISA 206佐剂是猪圆环病毒2型基因工程亚单位疫苗比较理想的佐剂。

甲醛灭活猪圆环病毒2型基因工程亚单位疫苗抗原液的最佳条件研究

为探究甲醛对猪圆环病毒2型基因工程亚单位疫苗抗原液最佳的灭活条件,本试验采用10%的甲醛溶液与猪圆环病毒2型基因工程亚单位疫苗抗原液混匀,使甲醛终浓度分别达到0.05%、0.07%、0.10%、0.15%、0.20%,在4~8℃下经过不同时间的灭活,通过对灭活液不同时间点的无菌检验及灭活检验,综合判定灭活效果。结果显示:猪圆环病毒2型基因工程亚单位疫苗抗原液在甲醛终浓度为0.07%时灭活72h,或甲醛终浓度为0.10%和0.15%时灭活60h,或甲醛终浓度为0.20%时灭活48h,均取得可靠的灭活效果。考虑到甲醛的毒副作用,笔者认为4~8℃下用0.07%甲醛终浓度灭活72h是比较理想的灭活条件。

禽白血病受体基因Tva在四个地方鸡种分布研究

为研究抗A/K亚群禽白血病病毒受体基因Tva易感基因与抗性等位基因在四个地方鸡种中的分布,研究采集斗鸡、仙居鸡、鹿苑鸡、藏鸡血样各25份进行Tva基因扩增后测序,并对测序结果进行基因分型统计。结果显示,所测鸡群体中全部存在抗性等位基因Tvar,且抗性等位基因在四个品种鸡群中呈现出不同程度的差异。等位基因Tva^(s)/Tva^(r4)在所测斗鸡群的频率为0.76/0.24,等位基因Tva^(s)/Tva^(r3)在所测藏鸡群中的频率为0.2/0.8,等位基因Tva^(s)/Tva^(r5)在所测鹿苑鸡群中的频率为0.48/0.52,等位基因Tva^(s)/Tva^(r4)/Tva^(r5)在所测仙居鸡群的频率为0.48/0.28/0.24。研究结果为进一步筛选与培育抗A/K亚群禽白血病病毒的鸡品系奠定基础。

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

Autoantibodies and hepatitis C virus genotypes in chronic hepatitis C patients in Estonia

AIM: To determine the prevalence of several autoantibodies in chronic hepatitis C patients, and to find out whether the pattern of autoantibodies was associated with hepatitis C virus (HCV) genotypes. METHODS: Sera from 90 consecutive patients with chronic hepatitis C were investigated on the presence of anti-nuclear (ANA), anti-mitochondrial (AMA), anti-smooth muscle (SMA), anti-liver-kidney microsomal type 1 (LKMA1), anti-parietal cell (PCA), anti-thyroid microsomal (TMA), and anti-reticulin (ARA) autoantibodies. The autoantibodies were identified by indirect immunofluorescence. HCV genotypes were determined by a restriction fragment length polymorphism analysis of the amplified 5' noncoding genome region. RESULTS: Forty-six (51.1%) patients were positive for at least one autoantibody. Various antibodies were presented as follows: ANA in 13 (14.4%) patients, SMA in 39 (43.3%), TMA in 2 (2.2%), and ARA in 1 (1.1%) patients. In 9 cases, sera were positive for two autoantibodies (ANA and SMA). AMA, PCA and LKMAI were not detected in the observed sera. HCV genotypes were distributed as follows: 1b in 66 (73.3%) patients, 3a in 18 (20.0%), and 2a in 6 (6.7%) patients. CONCLUSION: A high prevalence of ANA and SMA can be found in chronic hepatitis C patients. Autoantibodies are present at low titre (1:10) in most of the cases. Distribution of the autoantibodies show no differences in the sex groups and between patients infected with different HCV genotypes.

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.

Molecular epidemiology of respiratory syncytial virus

Objective To determine the epidemiologic pattern of subgroups A and B and genotypes of respiratory syncytial virus (RSV) during two noncontinuous epidemics during 1990-1991 and 1997-1998 in Beijing Methods Nasopharyngeal secretion (NPS) samples of RSV positive or RSV isolates tested by indirect immunofluorescence (IIF) assay were classified into subgroups A and B Isolates of RSV were divided into at least six different lineages, designated NP1 NP6, by restriction mapping of the N gene Np1, 3 and 6 were given by subgroup B isolates, while NP2, 4 and 5 were given by subgroup A isolates Strains of subgroup A were further subdivided into six lineages SHL1 SHL6 on the basis of the SH gene sequence SH lineages were closely related to each other and to NP1 NP6 Strains of SHL1, 3 and 4 were closely related and belonged to NP2, SHL2 and 6 to NP4, and SHL5 to NP5 Results Of 145 RSV NPS samples from the 1997-1998 epidemic, 83 (57 2%) were of subgroup B RSV positive, 62 (42 8%) of subgroup A RSV positive The rate of occurrence of subgroup A to B strains was about 1∶1 3 Two of 10 isolates during the epidemic were subgroup A strains, whereas 8 were subgroup B strains The rate of occurrence of subgroup A to B strains was 1∶4 Eight subgroup A strains of 10 isolates from the 1990-1991 epidemic were dominant; the proportion of subgroup A to B strains was 4∶1 With 10 RSV isolates in 1997-1998, all 2 subgroup A strains gave N gene fragment restriction pattern NP4, and fell into SH lineage SHL2, whereas 8 subgroup B strains all belonged to NP3 All 8 subgroup A isolates from the 1990-1991 epidemic gave pattern NP4, and fell into SHL2, while 2 subgroup B strains all belonged to NP3 The classification of subgroups A and B deduced from NP patterns corresponded to the definition of these subgroups by monoclonal antibodies Conclusions These observations confirm that subgroups A and B or multiple lineages of RSV co circulated in Beijing, but different genome types predominated each year Moreover, very similar viruses were isolated up to more than 5 years ago, indicating that despite apparent diversity of the subgroup A strains, the separate lineages might be relatively stable

Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China

Dear Editor,Zika virus（ZIKV）used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries（Enfissi et al.,2016）.Based on available clinical data,the symptoms in human infections with ZIKV are supposed to be similar to other arbovirus infections such as dengue,and characterized by fever,skin rashes,conjunctivitis,muscle and joint pain,malaise,and headache（Duffy et al.,2009）.