Determinants of pre-exposure rabies vaccination among foreign backpackers in Bangkok, Thailand

Important variations were observed regarding the proportion of backpackers seeking information about travel-associated diseases before departing for Thailand. The main determinants were nationality, reason for travel and age. Sources of information used by travelers varied substantially according to nationality. Moreover, significant differences were recorded regarding pre-exposure vaccination rates against rabies. Having British or Irish citizenship and seeking advice from travel clinic specialists or friends were the strongest and most significant determinants of rabies vaccination history. A significant relationship between vaccine cost and vaccination coverage was also evidenced.     

贵州及周边省市狂犬病病毒的分群和进化研究

目的 全面掌握贵州及周边省市狂犬病病毒(RABV)的遗传进化特征和分群特点,了解狂犬病疫苗株与流行株的分子遗传差异,为贵州狂犬病疫情的预防和控制提供指导依据.方法 搜索GenBank数据库中贵州及周边5个省市具有明确时间、地区及宿主信息的流行毒株,对其糖蛋白基因(G)全序列进行汇总,并结合本实验室获得的贵州毒株和国内现役疫苗株G序列,利用MEGA和BEAST软件分别进行系统进化和时间进化分析.结果 贵州及周边省市79株RABV均属于基因1型,9个类群的RABV流行株具有明显的地域特征,11株疫苗株分化为2个类群;RABV流行株与疫苗株G基因核苷酸和氨基酸同源性分别为0.827～0.999和0.713～0.998;贵州及周边省市RABV流行毒株的最近共同祖先(TMRCA)可追溯至150年前.结论 贵州及周边省市流行的RABV优势毒株群仍属于基因1型,但毒株经过多年流行已经出现了分化,有必要对流行毒株与疫苗株的遗传变异一致性进行深入研究.     

云南省保山市人间狂犬病调查及病毒分子生物学特征分析

目的 了解云南省保山市发生2例狂犬病患者的流行病学特点,根据病原分子生物学特征分析可能的传染来源.方法 采用狂犬病个案调查表进行流行病学调查,采集狂犬病死者脑组织标本,用直接免疫荧光试验(DFA)检测狂犬病病毒抗原,用RT-PCR法检测狂犬病病毒核酸,测定狂犬病病毒P、M和N基因全序列并根据其同源性比较及系统进化树进行分子流行病学分析.结果2006年7月保山市隆阳区发生1例人间狂犬病,2007年4月腾冲县发生1例狂犬病.2例患者犬伤暴露程度均为Ⅲ度.2例死亡病例均采集到脑组织(编号为CYN0601H和CYN0701H),经DFA和RT-PCR两种实验室方法检测确诊为狂犬病病毒阳性.CYN0601H和CYN0701H与国内外狂犬病病毒的P、M和N基因核苷酸和推导氨基酸序列同源性分析显示,CYN0601H和CYN0701H与泰国分离株的同源性最高.系统进化分析显示,2株病毒标本亲缘关系很近,同属于狂犬病病毒基因1型,且与泰国病毒株有较近的亲缘关系.结论从病原学和病毒分子水平证实云南省保山市2例患者均为狂犬病,保山市狂犬病病毒流行株可能为境外传人,应加强云南省边境地区狂犬病防制工作.     

河南省两株狂犬病毒核蛋白和糖蛋白基因的序列分析

目的分析河南省2株狂犬病毒核蛋白及糖蛋白的基因序列,了解狂犬病毒街毒株与人用及兽用狂犬病疫苗株间的差异。方法以免疫荧光法检测2004年采自河南省的100只犬脑标本,取阳性犬脑组织悬液接种鼠脑分离病毒。以RT-PCR法扩增病毒核蛋白及糖蛋白全基因,克隆测序后进行遗传学分析。结果分离到2株狂犬病毒,分别命名为Henan Hb1与Henan Sq1,序列分析表明2株病毒均为基因1型狂犬病毒,2株病毒的N基因与G基因核苷酸的同源性分别为99.3%和98.9%,氨基酸的同源性分别为98.7%和98.4%。2株病毒与CTN疫苗株同源性较高,N基因与G基因的核苷酸同源性分别为89.1%和85.6%～85.7%,氨基酸同源性分别为97.6%～98.0%和92.3%。与已知的基因1型狂犬病毒相比,2株病毒与印度尼西亚从犬中分离到的2株病毒同源性最高,N基因与G基因核苷酸同源性分别为92.1%～93.2%和91.9%～92.1%,氨基酸同源性分别为97.5%～98.6%和96.0%～96.2%。Henan Sq1株病毒在糖蛋白关键的毒力位点333位由W替换了R。系统发育分析表明Henan Hb1与Henan Sq1与印度尼西亚株、疫苗株CTN、中国分离株,以及泰国与马来西亚分离株进化关系最近,而与疫苗株3aG、PV、ERA及攻击毒CVS株等进化关系较远。结论2株狂犬病毒是基因1型狂犬病毒,但无论是N基因还是G基因的核苷酸序列,以及推导出来的氨基酸序列与已知的1型狂犬病毒株及疫苗株均有一定的差异。     

广西狂犬病病毒分子流行病学研究

目的分析广西狂犬病毒的分布和来源,从病原学角度分析广西狂犬病疫情高发的原因。方法2005年9月~2006年4月在广西玉林、贵港、柳州、来宾、南宁和河池等6市共收集健康犬脑标本1 352份,用直接免疫荧光法(DFA)和RT-PCR法检测狂犬病毒,其中22份标本完成狂犬病毒N基因编码区下游720bp核苷酸序列的测定。结果用DFA法检测出阳性标本160份,阳性率为11.83%,RT-PCR检测出阳性标本26份,阳性率1.9%,对其中22份标本完成N基因编码区下游720bp核苷酸序列的测定,核苷酸序列同源性为87.8%~100%,推导氨基酸序列的同源性为97.5%~99.6%,表明广西病毒标本N基因核苷酸序列的变异主要是同义突变。种系发生分析显示,广西病毒标本分为A、B、C群,各群分布范围不同,具有明显地域性特征;中国存在的3群狂犬病毒(CHINA-1、CHINA-2和CHINA-3群)目前在广西均有分布。广西流行的3群病毒来源各不相同:A群可能来源于与广西相邻的湖南、贵州;B群可能由广西北部省区传入;C群是在广西境内循环的毒株。结论广西境内普遍存在狂犬病毒感染犬只,并有外省狂犬病毒的传入,可能是近年广西狂犬病疫情上升的重要原因。     

Assessment of inactivated human rabies vaccines: biochemical characterization and genetic identification of virus strains

The World Health Organization (WHO) recommends the periodic evaluation of the purity of the cell lines used in the production of rabies vaccines, as well as the antigenic identity of the virus strains. Here, we analyzed seventeen marketed inactivated human rabies virus vaccines for viral and non-viral proteins by SDS-PAGE and Coomassie/silver staining. Mass spectrometric analysis of an abundant 60-70 kDa signal indicated that in most vaccines serum albumin of human origin (HSA) was the major component. Quantification of HSA in the vaccines revealed a mean concentration of 22 mg HSA/dose in all tested PVRV (purified vero cell rabies vaccine), HDCV (human diploid cell rabies vaccine) and PHK (primary hamster kidney) vaccines. In contrast, 1000-fold lower HSA levels and no HSA were detected in PCECV (purified chick embryo cell-culture vaccine) and PDEV (duck embryo rabies vaccine), respectively. Western blot analyses further confirmed a high bias in the HSA content, whereas the virus protein levels were rather similar in all tested vaccines. In addition, the vaccine viruses were sequenced within the N- and G-genes to identify the strain. In the majority of sequenced vaccines, the declared vaccine strain was confirmed. However, some discrepancies in the genetic identification were observed, supporting WHO's recommendation for the molecular characterization of vaccine seed strains. This research highlights the variation in purity found between different human rabies virus vaccines, and suggests that further research is needed to establish the impact non-active components have on the potency of such vaccines.     

狂犬病病毒浙江街毒株糖蛋白基因序列分析

目的 测定狂犬病病毒浙江株(鼬獾源和犬源)糖蛋白(GP)基因组全序列,从分子水平比较狂犬病病毒浙江株与其他地区代表性街毒株和疫苗株GP之间的差异.方法 乳鼠接种分离狂犬病病毒,RT-PCR反应测定狂犬病病毒浙江株GP基因核苷酸序列,并进行序列和编码蛋白的比较分析.结果测序获得浙江5株鼬獾源狂犬病病毒和9株犬源狂犬病病毒GP基因序列,全长1575个核苷酸,编码524个氨基酸.浙江病毒株与其他地区街毒株、疫苗株核苷酸和氨基酸序列相似性在82.3%～99.9%和85.1%～99.8%之间,种系分析显示浙汀株均为基因1型,GP编码蛋白无重组,主要抗原位点未出现较大的变异.结论 对GP基因一级结构综合分析表明,在某些区域存在优势抗原表位的可能性较大,可能出现潜在的蛋白质抗原决定簇,浙江株GP基因变异较小,与国内其他地区流行的代表性街毒株相似,但高于其他疫苗株,在基因结构及种系进化关系上存在一定的距离.     

Alanine scanning of the rabies virus glycoprotein antigenic site III using recombinant rabies virus: Implication for post-exposure treatment

The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination.     

浙江省慈溪市犬类狂犬病病毒携带状况调查及变异研究

目的研究犬类狂犬病病毒的携带状况及变异,为正确有效控制狂犬病疫情提供依据。方法根据狂犬病疫情分布,用分层采样法,采集相关地区犬脑51只,用免疫荧光法、夹心酶联吸附法、小鼠颅内接种试验法及RT-PCR进行检测。分离狂犬病病毒并进行N基因测序。结果在流浪犬只中发现狂犬病毒株(命名CXs)。流浪犬带毒率7.69%,CXs与疫苗株和固定毒株的NJ进化树比较,发现与WZO(H)株100%同源,与狂犬病毒固定毒株相比更接近CTN株(该地区使用的疫苗株)。结论当前使用的狂犬疫苗用于预防狂犬病是可靠的。经狂犬病流行特征和暴露后免疫预防处理分析,加强犬只的管理、免疫,暴露后采用规范清创,正确使用狂犬疫苗,合理使用狂犬免疫球蛋白(或血清)仍是当前防治狂犬病需遵循的原则。     

乙型肝炎病毒基因型和血清亚型在我国部分地区的分布及其特点

目的 研究乙型肝炎病毒（hepatitis B virus,HBV）基因型和血清亚型的分布特点及其基因的相关性。方法 湖南、广西、河南和河北4省（区）25个县（市）280例慢性HBV携带者,应用聚合酶链反应（PCR）扩增和脱氧核糖核酸（DNA）序列分析,确定HBV基因型和血清亚型。结果 HBV基因型B、C和D均有分布,分别占29.3%、67.9%和2.9%,其中B和C为优势基因型。adr、adw2、ayr、aywl、ayw2和ayw3等6种血清亚型均有分布,其中adr和adw2为优势血清亚型,分别占64.3%和31.4%。基因型B与adw2血清亚型,基因型C与adr血清亚型有非常密切的基因相关性。280例HBV携带者表面抗原基因序列每100个核苷酸的平均置换频数为2.94。基因型B(adw2血清亚型）毒株核苷酸置换频数为5.63(5.48）,而基因型C（adr血清亚型）仅为1.6(1.51）。结论 HBV基因型和血清亚型的分布均有明显的地区性;基因型B及相对应的adw2血清亚型毒株的基因可变性明显高于基因型C及相应的adr血清亚型。     

Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa

Western BTV-1 emerged in the Mediterranean basin in 2006 and it has since been isolated in southern and northern European countries. Six BTV-1 strains isolated from infected sheep in Italy between 2006 and 2013 and a BTV-1 strain isolated from an infected sheep in Tunisia in 2011 were fully sequenced. The seven strains were shown to be nearly identical in each gene segment. The Seg-2 sequences of the BTV-1 strains group according to the year of isolation reflecting the time of BTV incursions in Italy. Combined results suggest that BTV-1 strains isolated in Sardinia, Sicily and mainland Italy in 2012 and 2013 have a direct northern African origin. The Italian strains originated from a strain closely related to a BTV-1 strain isolated in Tunisia in 2011. Better surveillance programs with northern and sub-Saharan African countries should be implemented making the control of spread of BTV easier and effective.     

Hepatitis A virus: Host interactions,molecular epidemiology and evolution

Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions.     

Rationale and support for a One Health program for canine vaccination as the most cost-effective means of controlling zoonotic rabies in endemic settings

Although dog vaccination has been demonstrated to reduce and eliminate rabies in humans, during meetings there are often calls for further pilot studies. The assembled data proves that a widespread approach is now required. While zoonotic rabies has a minimal presence in developed nations, it is endemic throughout most of Asia and Africa, where it is considered to be a neglected tropical disease. In these areas, rabies causes an estimated annual mortality of at least 55,000 human deaths. Worldwide rabid dogs are the source of the vast majority of human rabies exposures. The World Health Organization (WHO), the Food and Agriculture Organization (FAO) of the United Nations and the World Organization for Animal Health (OIE) advocate a collaborative One Health approach involving human public health and veterinary agencies, with mass canine vaccination programs in endemic areas being the mainstay of strategies to eliminate dog-mediated human rabies. While post-exposure prophylaxis (PEP) is effective in preventing deaths in people exposed to rabies, it is comparatively expensive and has little impact on the canine reservoir that is the primary source of zoonotic rabies. Indiscriminate culling of the dog population is expensive and there is little evidence that it is effective in controlling rabies in non-island locations. Mass canine vaccination programs using a One Health framework that achieves a minimum 70% vaccination coverage during annual campaigns have proven to be cost-effective in controlling zoonotic rabies in endemic, resource-poor regions. Case studies, such as in Tanzania and Bhutan, illustrate how an approach based on mass canine rabies vaccination has effectively reduced both canine and human rabies to minimal levels. The multiple benefits of mass canine rabies vaccination in these cases included eliminating rabies in the domestic dog reservoirs, eliminating human rabies cases, and decreasing the rabies economic burden by reducing expenditures on PEP.     

An assessment of shedding with the oral rabies virus vaccine strain SPBN GASGAS in target and non-target species

A safety requirement for live vaccines is investigating possible shedding in recipients since the presence of replication competent vaccine in secretions could result in direct and indirect horizontal transmission. This is especially relevant for oral rabies vaccine baits that are deliberately distributed into the environment. In the current study, survival of an oral rabies virus vaccine, SPBN GASGAS, was examined in excretions from different target and non-target species; red fox, raccoon dog, small Indian mongoose, raccoon, striped skunk, domestic dog, domestic cat and domestic pig. Saliva – and (pooled) fecal samples collected at different time points after oral administration of the vaccine strain were examined for the presence of viral RNA (rt-PCR). All PCR-positive and a subset of PCR-negative samples were subsequently investigated for the presence of infectious virus by isolation in cell culture (RTCIT). Up to 7 days post vaccine administration viral RNA could be detected in 50 of 758 fecal samples but no infectious virus was detected in any of the examined PCR-positive fecal samples. In contrast, RNA-fragments were detected in 248 of 1053 saliva swabs for an extended period (up to 10 days) after vaccine administration, but viable virus was only present during the first hours post vaccine administration in 38 samples. No infectious vaccine virus was isolated in saliva swabs taken 24 h or more after vaccine administration. Hence, no active shedding of the vaccine virus SPBN GASGAS after oral administration occurred and the virus isolated during the initial hours was material originally administered and not a result of virus replication within the host. Thus, potential horizontal transmission of this vaccine virus is limited to a short period directly after vaccine bait uptake. It can be concluded that the environmental risks associated with shedding after distributing vaccine baits containing SPBN GASGAS are negligible.     

北京地区2006年B型流感病毒HAl基因特征分析

近期在北京地区的流感监测中发现B型流感病毒经常引起局部暴发,因此我们对最近分离到的B型流感病毒重要抗原血凝素蛋白重链区(HA1)进行了核苷酸和氨基酸序列分析,结果报告如下。1．流感病毒分离鉴定:2005-2006年共分离到流感病毒     

Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein

We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection.     

广西狂犬病病毒与狂犬病疫苗株N基因序列分析

目的了解广西狂犬病病毒（RABV）街毒株与疫苗株N基因序列的差异,为疫苗使用提供理论依据。方法采用直接免疫荧光法（DFA）和巢式RT—PCR法检测,测定病毒N基因序列全长,同时根据N基因序列同源性及系统进化树比较,分析广西近年流行的RABV与国内外10株疫苗代表株N基因序列之间的差异。结果DFA法阳性率为8．84％（350／3961）,RT—PCR法检测阳性率1．29％（51／3961）,获得N基因全序15份;15份N基因核苷酸与氨基酸序列同源性为89．40％。100％和98．40％~99．80％;广西15份（RABV）与国内外10株疫苗代表株N基因核苷酸和氨基酸序列同源性分别为85．70％～99．70％和94．90％～99．80％,与中国CTN疫苗株N基因的核苷酸和氨基酸同源性最高,分别为89．60％,99．70％和98．40％。99．80％。N基因系统发生树分为两大分支,15份（RABV）与中国人用CTN株构成第一分支,其余的疫苗毒株构成第二分支。结论广西犬间流行的RABV与中国人用CTN疫苗株N基因序列同源性最高、亲缘关系最近,在广西地区使用CTN疫苗株效果可能较好。     

湖南省2008-2009年狂犬病病原学监测及病毒基因特征分析

目的 分析2008-2009年湖南省狂犬病病原学监测结果及新分离病毒的N基因分子特征.方法 采用直接免疫荧光法(DFA)、巢式PCR检测狂犬病监测标本,阳性标本应用ABI3730测序仪对N基因进行全序列测序;运用生物信息学方法分析N基因特征及序列同源性,构建系统进化树,分析新分离病毒株遗传特征并与以往分离株比较.结果 1451份监测犬脑组织标本中DFA初筛阳性31份,阳性率为2.14％;31份DFA阳性标本经巢式PCR复核,17份阳性,阳性率为1.17％;巢式PCR检测疑似狂犬病病例唾液、脑脊液、血清及尿液标本56份,3份阳性,阳性率为5.36％;新分离的阳性株与巴斯德株进行N基因序列比较,核苷酸和氨基酸同源性均在87.2％～ 87.9％之间;成功构建系统进化树,新分离的20株病毒全部属于基因Ⅰ型.新分离病毒株与湖南省内、邻省和国际株比较存在不同的亲缘关系.结论 湖南省狂犬病病例及犬携带病毒的情况较为稳定,流行株仍为基因Ⅰ型,未发生变异.     

Spirituality within the family and the prevention of health risk behavior among adolescents in Bangkok, Thailand

This study investigates the influences of a family's spiritual beliefs and practices on substance use and sexual risk behaviors among young adolescents 13-14 years old in Bangkok, Thailand. Independent predictor variables are the parents' and teens' spiritual beliefs and practices in Buddhism and parental monitoring behaviors. The study uses data from the 2007 Baseline Survey of the Thai Family Matters Project, which adapted a U.S. based family prevention program for Thai culture. A representative sample of 420 pairs of parents and teens from the Bangkok metropolitan area was recruited to participate in the study. Structural equation models indicate that positive direct and indirect associations of the spirituality of parents and teens within a family and the prevention of adolescent risk behaviors are significant and consistent.