1996-2014年中国HIV-1毒株CRF01_AE亚型传播簇和传播网络研究

目的 了解我国HIV-1毒株CRF01_AE亚型在省内和省际的传播规律和风险因素,为实施精准干预提供参考依据。方法 收集我国19个省份1996-2014年已有的2 094条CRF01_AE pol区基因序列,利用PhyML 3.0软件构建系统进化树,确定传播簇,利用Cytoscape 3.6.0软件构建传播网络,结合背景信息分析传播风险。结果 发现82个传播簇,包含255条序列（12.18%,255/2 094）,省内传播簇数量和包含序列数（61个,173条）明显多于跨省传播簇（21个,82条）。传播簇中男男性传播人群的成簇比例随时间上升趋势明显,由1996-2008年的2.41%（2/83）上升为2013-2014年的23.61%（72/305）（χ²=27.800,df=1,P=0.000）。跨省传播簇的男男性传播人群比例明显高于省内传播簇,由1996-2008年的0.67%（2/297）上升为2013-2014年的6.36%（30/472）,具有随时间的上升趋势（χ²=20.276,df=1,P=0.000）。跨省传播簇中男男性传播的比例（86.59%,71/82）明显高于省内传播簇（56.65%,98/173）,差异有统计学意义（χ²=22.792,P=0.000）。含2种及以上传播途径的跨省传播簇的比例（33.33%,7/21）明显高于省内传播簇（13.11%,8/61）,差异有统计学意义（χ²=4.273,P=0.039）。传播网络分析发现,跨省传播簇内高传播风险人群比例（51.22%,42/82）明显高于省内传播簇（26.59%,46/173）,差异有统计学意义（χ²=14.932,P=0.000）。跨省传播簇以男男性传播人群为主。结论 我国HIV-1毒株CRF01_AE亚型存在复杂的传播网络,跨省传播簇快速增长,其中高风险传播者对HIV-1亚型的大范围传播起到重要作用,应深入进行传播网络研究以指导精准干预。     

重要呼吸道病毒病原的多重RT-PCR检测

目的 建立一种可同时快速检测引起人呼吸道感染的重要病毒病原的检测方法。方法 通过对PCR反应条件的优化，建立同时检测甲型流感病毒（IA）、呼吸道合胞病毒（RSV）和SARS冠状病毒（SARS CoV）的多重RT-PCR方法。结果 该法可同时或分别扩增IA258bp、RSV348bp和SARS-CoV438bp的基因片段。检测的敏感性分别为10^17TCID50／ml、10TCID30／ml和10^1.5。TCID50／ml。采用相同的反应体系和条件，分别对其他7种呼吸道病毒（包括乙型流感病毒，副流感病毒2型，柯萨奇病毒B3和B5血清型及腺病毒2、3和7血清型）进行扩增，均未检测出扩增产物。结论此种多重RT-PCR法可在6h内完成，适用于三种重要呼吸道病毒的快速甄别。     

Genomic epidemiological investigation of a Streptococcus suis outbreak in Guangxi,China, 2016

In June 2016, a Streptococcus suis outbreak occurred in Guangxi, China. We determined the genetic characteristics of six clinically isolated strains by serotyping, PCR, and whole-genome sequencing, performing genome epidemiology analysis on these and 961 public available S. suis genomes. We also classified the first sequence type ST665 human case. Sporadic and outbreak cases were distinguished by whole-genome sequencing and phylogenomics. This approach could help to prevent and control S. suis epidemics in Guangxi and the wider region.     

Live attenuated Salmonella enterica serovar Choleraesuis vector delivering a conserved surface protein enolase induces high and broad protection against Streptococcus suis serotypes 2, 7, and 9 in mice

Streptococcus suis, a major zoonotic pathogen in swine, can be classified into 35 serotypes. However, no universal vaccine against the multiple serotypes of S. suis is available, though some studies have shown homologous protection. Hence, developing an effective universal vaccine to protect pigs against multiple S. suis serotypes is necessary, or at the very least, to protect pigs against diseases caused by the dominant pathogenic serotypes. Enolase, a highly conserved surface protein, is present in all of the described S. suis serotypes. rSC0016 is an improved recombinant attenuated S. Choleraesuis vaccine vector, combining a sopB mutation with regulated delayed systems, achieving an adequate balance between host safety and immunogenicity. In order to develop a universal vaccine against the multiple serotypes of S. suis, a novel recombinant vaccine strain rSC0016 that carries a heterologous antigen enolase was developed in this study. According, it was found that the recombinant vaccine strain rSC0016(pS-Enolase) exhibited better colonization compared to the vaccine control strain rSC0018(pYA3493). In addition, a mouse model immunized with the strain rSC0016(pS-Enolase) elicited significant IgG antibody responses against both enolase and Salmonella antigens, while inducing good mucosal, humoral, and cellular immune responses against enolase. Finally, immunization with rSC0016(pS-Enolase) was shown to confer 100%, 80%, and 100% protection against the serotypes of SS2, SS7, and SS9, respectively, and significantly reduced histopathological lesions in mice. Overall, this study provides a promising universal vaccine candidate for use against the multiple serotypes of S. suis.     

Hazardous alcohol consumption and aging synergistically increase the risk of death in patients with severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case fatality rate (CFR). Alcohol consumption which impairs host immunity and contributes to tissue damage in a variety of organs may be a predisposing factor of fatal outcome in SFTS. We aimed to determine the role of alcohol consumption on the fatal outcome of SFTS. Patients with laboratory-diagnosed SFTS who were admitted to the Jinan Infectious Disease Hospital, Jinan, China, between January 2011 and November 2018 were evaluated. Demographic, clinical, and laboratory data were recorded. Alcohol consumption was evaluated. The association between a fatal outcome and each demographic, clinical, and laboratory variable with alcohol consumption was assessed. A total of 694 patients with SFTS were identified during the study period. The overall CFR was 20.9 % (95 % CI: 17.9 %–23.9 %). The CFR in non/light drinkers (0−98 g/week) and moderate/heavy drinkers (>98 g/week) was 18.3 % and 35.6 %, respectively (P < 0.001). In age>60 years patients, the overall CFR in moderate/heavy drinker groups were as high as 53.4 % (95 % CI:40.2 %–66.7 %). Comparing to the age≤60y and non/light drinkers, age>60y and moderate/heavy drinkers was associated with increased risk of death with an odds ratio (95 % CI) of 9.9 (5.1–19.1). The interaction between age>60 and alcohol consumption was a significant determinant for death in both genders (F=10.18, P = 0.001). The clinical manifestation, laboratory parameters, and organ injury were significantly extensive and severe in moderate and heavy drinkers. In conclusion, hazardous alcohol consumption and aging synergistically increase the risk of death in patients with SFTS. In SFTS endemic areas, it is important for older individuals to minimize the exposure risks and abstain from alcohol.     

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein–Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated ‘nontypeable’ strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent.     

荧光定量PCR快速检测金黄色葡萄球菌方法的建立

目的利用SmartCycler系统建立一种简便、快速的荧光定量PCR检测金黄色葡萄球菌（Staphylococcus aureus,简称SAU）的方法。方法根据SAU特异nuc基因设计引物和探针,建立检测体系,并在体系中加入内质控IAC,通过另一个标记不同荧光基团的TaqMan探针来监测IAC。用SAU6538株污染饮用水和牛奶模拟实际样本,以评价体系的性能。结果以含nuc片段的线性质粒为模板,反应体系的灵敏度为5拷贝/μl;以6538株基因组DNA为模板,最低检测限为10fg/μl;用涂平板法计数并10倍梯度稀释的菌液提取DNA为模板,最低检测限为50cfu/ml。建立的定量标准曲线的循环域值（Ct）与模板拷贝数呈良好的线性关系（r2≥0.998）,PCR效率E≥96.7%。用SAU6538污染饮用水和牛奶模拟实际样本,灵敏度可达5×102cfu/25ml样本。结论所建立的nuc-IAC荧光定量PCR具有内质控IAC,既能定量检测食物是否被污染,又能监测PCR体系,防止假阴性发生或低估病原菌污染量。     

呼吸道合胞病毒的分离鉴定及其致病力的初步评价

目的分离鉴定临床呼吸道合胞病毒(respiratory syncytial virus,RSV),评价其致病力,为疫苗评价及致病机制的研究提供实验材料。方法收集临床呼吸道感染重症患儿的鼻咽分泌物样本进行RT-PCR初步鉴定,通过病毒培养,空斑纯化,电镜观察及病毒滴度测定,筛选出1株呼吸道合胞病毒候选株,经全基因组测序后命名为BJ016。选用14日龄C57乳鼠,每组26只,采用BJ016为实验组,PBS为对照组,分别对小鼠滴鼻感染,30μl/只。连续14日监测2组小鼠体质量变化及死亡率,肺组织的病毒载量、湿干比,HE染色观察肺病理变化并计数炎性细胞总量,同时采用CBA测定血清中细胞因子的变化。结果 100份临床呼吸道感染重症患儿鼻咽分泌物样本中,RT-PCR鉴定为RSV A亚型阳性共21例,经病毒分离培养、空斑纯化、电镜观察及RT-PCR鉴定后得到16株RSV临床分离株,通过TCID50测定病毒滴度筛选出1株滴度为106.5TCID50/ml的候选株,基因组序列分析显示其为NA1基因型,命名为BJ016。BJ016感染14日龄C57乳鼠后造成肺损伤,在第5日达到发病高峰时测定病载、湿干比、病理检测并计数炎性细胞,实验组较对照组具有明显统计学差异,血清中MCP-1、IL-6、G-CSF及MIP-1α均显著性升高。结论成功分离出1株能致死14日龄C57乳鼠的RSV病毒株并初步评价了其致病力,为疫苗评价及RSV致病机制的研究提供了良好的实验材料。