尼帕病毒病及其病原的研究进展

概述了尼帕病毒病在国外的流行状况、尼帕病毒（Nipah virus，NiV）的变异及我国的研究现状，着重阐述了近年来在其基础病毒学、诊断技术及防治方面的研究进展，主要包括病毒蛋白的功能及其相互作用、受体的发现、动物模型、荧光PCR及ELISA诊断技术、抑制NiV感染及治疗等方面的研究成果，为该病的深入研究提供了参考。     

Experimental inoculation study indicates swine as a potential host for Hendra virus

Hendra virus (HeV) is a zoonotic virus from the family Paramyxoviridae causing fatal disease in humans and horses. Five-week-old Landrace pigs and 5-month-old Gottingen minipigs were inoculated with approximately 10⁷ plaque forming units per animal. In addition to fever and depression exhibited in all infected pigs, one of the two Landrace pigs developed respiratory signs at 5 days post-inoculation (dpi) and one of the Gottingen minipigs developed respiratory signs at 5 dpi and mild neurological signs at 7 dpi. Virus was detected in all infected pigs at 2–5 dpi from oral, nasal, and rectal swabs and at 3–5 dpi from ocular swabs by real-time RT-PCR targeting the HeV M gene. Virus titers in nasal swab samples were as high as 10^4.6 TCID₅₀/mL. The viral RNA was mainly distributed in tissues from respiratory and lymphoid systems at an early stage of infection and the presence of virus was confirmed by virus isolation. Pathological changes and immunohistochemical staining for viral antigen were consistent with the tissue distribution of the virus. This new finding indicates that pigs are susceptible to HeV infections and could potentially play a role as an intermediate host in transmission to humans.     

Serological evidence of Coxiella burnetii infection in beef cattle in Queensland

Background Queensland has the highest incidence of Q fever in Australia. The aim of this study was to undertake a cross‐sectional seroprevalence survey of Coxiella burnetii, the causative agent of Q fever, in beef cattle in Queensland. Methods Serum samples were tested by ELISA for both phase II and phase I antigens of the organism using an Australian isolate. Blood samples were collected at an abattoir that processes beef cattle originating from northern and north‐western Queensland, in addition to blood samples taken from beef cattle across Queensland as part of a second survey. Results Seropositivity was 16.8% (95% confidence interval 16.7–16.8%). Conclusion Evidence of C. burnetii infection in beef cattle has public health implications for occupational exposure of primary producers and veterinarians and for the proximity of beef cattle properties to residential areas in regional Queensland. This study is the first known investigation of C. burnetii seroprevalence in beef cattle in Queensland and the first known use of an Australian C. burnetii isolate for screening using both phase II and phase I antigens.     

Animal models of henipavirus infection: A review

Hendra virus (HeV) and Nipah virus (NiV) form a separate genus Henipavirus within the family Paramyxoviridae, and are classified as biosafety level four pathogens due to their high case fatality rate following human infection and because of the lack of effective vaccines or therapy. Both viruses emerged from their natural reservoir during the last decade of the 20th century, causing severe disease in humans, horses and swine, and infecting a number of other mammalian species. The current review summarises current published data relating to experimental infection of small and large animals, including the natural reservoir species, the Pteropus bat, with HeV or NiV. Susceptibility to infection and virus distribution in the individual species is discussed, along with the pathogenesis, pathological changes, and potential routes of transmission.     

尼帕病毒和亨得拉病毒核蛋白单克隆抗体的制备与鉴定

尼帕病毒（Nipahvirus，NiV）和亨得拉病毒（Hendravirus，HeV）是近年来出现的2种新的高致病性副粘病毒，在我国尚未发现。为防范2种病毒在我国的出现，本研究开展了前瞻性工作，成功研制了针对2种病毒核蛋白（N）的单克隆抗体，可用于病毒监测与诊断。首先利用大肠杆菌表达的2种病毒N蛋白免疫BALB／c小鼠，细胞融合后应用间接免疫荧光的方法对杂交瘤细胞克隆进行筛选，获得了5株N蛋白特异单抗。单抗腹水的抗体效价均超过2×10^5，培养上清抗体效价1：64～1：256。Western—blot和间接免疫荧光试验证明，5株单抗均特异针对N蛋白，其中1株（H4D11）只与HeVN蛋白反应，而不与NiVN蛋白反应，表明它具有鉴别2种病毒的能力。所研制的单抗对建立2种病毒的检测技术，用于动物监测，以防范2种新发传染病在我国流行具有重要意义。     

Serological evidence for exposure to bovine viral diarrhoea virus in sheep co-grazed with beef cattle in New Zealand

ABSTRACT

Aims: To determine whether sheep that co-grazed with cattle that were suspected to be positive for bovine viral diarrhoea (BVD) virus had serological evidence of exposure to the virus.

Methods: Eighteen commercial farms that routinely co-grazed cattle and sheep in the same paddocks were recruited through purposive sampling. The recruiting veterinarians identified nine farms with cattle herds that were known or highly suspected to be positive for BVD and nine farms that were considered to be free of BVD. Blood samples were taken from 15 ewes aged 1 year on each farm and samples were submitted to a commercial diagnostic laboratory to test for antibodies against pestiviruses using an ELISA. All samples that were positive were then tested using a virus neutralisation test (VNT)for antibodies against BVD virus.

Results: Of the 270 blood samples, 17 were positive for pestivirus antibodies by ELISA and these originated from two farms that were known or suspected to have BVD virus-positive cattle. None of the samples from the nine flocks co-grazed with cattle herds that were known or suspected to be BVD virus-negative were positive for pestivirus antibodies. Within the two positive farms, 2/15 samples from the first farm and 15/15 samples from the second farm were antibody-positive. When the 17 positive blood samples were submitted for VNT, all 15 samples from the second farm tested positive for BVD virus antibodies with the highest titre being 1:512.

Conclusions and clinical relevance: In this small sample of New Zealand sheep and beef farms with suspected BVD infection in cattle, there was evidence of pestivirus exposure in co-grazed sheep. Although we were unable to confirm the origin of the exposure in these sheep, these findings highlight that farmers who are trying to eradicate BVD from their cattle should be mindful that the infection may also be circulating in sheep, and both populations should be considered a possible risk to each other for generating transient and persistent infections. Further work is needed to estimate the true prevalence of New Zealand sheep flocks that are affected by BVD and the associated economic impacts.     

Intangible and Economic Impacts of Hendra Virus Prevention Strategies

Hendra virus (HeV), a potentially fatal zoonotic disease spread by flying foxes, to date has always infected humans via a spillover event from equine HeV infection. In a theoretical case study, we compared the impacts of two different HeV prevention strategies – vaccination and flying fox roost removal – using a recently developed framework that considers different stakeholder group perspectives. The perspectives of the four selected stakeholder groups regarding intangibles were inferred from public discussions and coverage in the media. For all stakeholder groups, the option to vaccinate horses was found to add value to the economic results when the intangible impacts were included in the analysis, while the option for roost removal unanimously detracted from economic analysis value when the intangible impacts were included. Both the mean and median stakeholder‐adjusted value ratios (2.25 and 2.12, respectively) for vaccination were inflated when intangible impacts were included, by value‐adding to the results of a traditional economic analysis. In the roost removal strategy, these ratios (1.19 and 1.16, respectively) were deflated when intangible impacts were included. Results of this theoretical study suggest that the inclusion of intangible impacts promotes the value of a two‐dose initial vaccination protocol using a subunit vaccination considered to offer complete protection for horses, as a strategy to control HeV, whereas roost removal becomes an even more costly strategy. Outcome of the analysis is particularly sensitive to the intangible value placed on human health. Further evaluation – via sociological methods – of values placed on intangibles by various stakeholder groups is warranted.     

Hendra virus in Queensland,Australia, during the winter of 2011: Veterinarians on the path to better management strategies

Following the emergence of Hendra virus (HeV), private veterinarians have had to adopt additional infection control strategies to manage this zoonosis. Between 1994 and 2010, seven people became infected with HeV, four fatally. All infected people were at a higher risk of exposure from contact with horses as they were either veterinary personnel, assisting veterinarians, or working in the horse industry. The management of emerging zoonoses is best approached from a One Health perspective as it benefits biosecurity as well as a public health, including the health of those most at risk, in this case private veterinarians. In 2011 we conducted a cross-sectional study of private veterinarians registered in Queensland and providing veterinary services to horses. The aim of this study was to gauge if participants had adopted recommendations for improved infection control, including the use of personal protective equipment (PPE), and the development of HeV specific management strategies during the winter of 2011. A majority of participants worked in practices that had a formal HeV management plan, mostly based on the perusal of official guidelines and an HeV field kit. The use of PPE increased as the health status of an equine patient decreased, demonstrating that many participants evaluated the risk of exposure to HeV appropriately; while others remained at risk of HeV infection by not using the appropriate PPE even when attending a sick horse. This study took place after Biosecurity Queensland had sent a comprehensive package about HeV management to all private veterinarians working in Queensland. However, those who had previous HeV experience through the management of suspected cases or had attended a HeV specific professional education programme in the previous 12 months were more likely to use PPE than those who had not. This may indicate that for private veterinarians in Queensland personal experience and face-to-face professional education sessions may be more effective in the improvement of HeV management than passive education via information packages. The role of different education pathways in the sustainable adoption of veterinary infection control measures should be further investigated.     

亨得拉病毒核蛋白基因(N)在大肠杆菌的高效表达与反应原性鉴定

从引进的含亨得拉病毒（Hendra virus，HeV）基因的克隆质粒中扩增得到该病毒核蛋白基因（N），将其克隆到pET-28a（＋）原核表达栽体，转化大肠杆菌后成功地表达了N蛋白，SDS-PAGE电泳分析结果表明目的基因在JM109（DE3）大肠杆菌菌株中得到了良好的表达，目的蛋白量占菌体总蛋白的14．6％。Western-blotting检测表明，表达的蛋白可以被兔抗亨得拉全病毒阳性血清识别，表明该蛋白具有良好的反应性。该研究结果可进一步应用于诊断试剂和单克隆抗体的制备以及流行病学调查，以防范该病在我国的流行。     

Serological evidence of Coxiella burnetii infection in dogs in a regional centre

OBJECTIVE Investigate the seroprevalence of the causative agent of Q fever, Coxiella burnetii in domestic dogs in the Townsville region, North Queensland, Australia. METHOD Blood samples were collected from dogs attending veterinary clinics for routine procedures. RESULTS An overall seropositivity of 21.8% (95% confidence interval (CI) 21.6-22.1%) was observed. A retrospective study of samples collected in the same region during 1984-85 was also performed, with an overall seropositivity of 16.0% (95% CI 15.9-16.2). CONCLUSION Evidence of C. burnetii infection in domestic dogs may have public health implications for dog owners, as well as veterinarians because of occupational exposure. This study is the first known investigation of C. burnetii seroprevalence in dogs in Queensland.     

尼帕病毒受体结合糖蛋白在哺乳动物细胞中的可溶性表达和纯化及小鼠抗血清制备

参照GenBank中尼帕病毒受体结合蛋白(G蛋白)编码序列,使用DNAStar软件对各基因型尼帕病毒G蛋白进行核苷酸、氨基酸同源性分析,并绘制进化树,选择代表性毒株(NCBI登录号:AF212302.2),截取合成G蛋白头部结构域(sG)的核苷酸序列,连接至pcDNA3.1(+)真核表达载体上,构建为重组质粒pcDNA...     

Serological evidence for exposure to avian influenza viruses within poultry workers in southern China

The risk of infection with avian influenza viruses for poultry workers is relatively unknown in China, and study results are often biased by the notification of only the severe human cases. Protein microarray was used to detect binding antibodies to 13 different haemagglutinin (HA1‐part) antigens of avian influenza A(H5N1), A(H7N7), A(H7N9) and A(H9N2) viruses, in serum samples from poultry workers and healthy blood donors collected in the course of 3 years in Guangdong Province, China. Significantly higher antibody titre levels were detected in poultry workers when compared to blood donors for the most recent H5 and H9 strains tested. These differences were most pronounced in younger age groups for antigens from older strains, but were observed in all age groups for the recent H5 and H9 antigens. For the H7 strains tested, only poultry workers from two retail live poultry markets had significantly higher antibody titres compared to blood donors.     

No Evidence of Hendra Virus Infection in the Australian Flying‐fox Ectoparasite Genus Cyclopodia

Hendra virus (HeV) causes potentially fatal respiratory and/or neurological disease in both horses and humans. Although Australian flying‐foxes of the genus Pteropus have been identified as reservoir hosts, the precise mechanism of HeV transmission has yet to be elucidated. To date, there has been limited investigation into the role of haematophagous insects as vectors of HeV. This mode of transmission is particularly relevant because Australian flying‐foxes host the bat‐specific blood‐feeding ectoparasites of the genus Cyclopodia (Diptera: Nycteribiidae), also known as bat flies. Using molecular detection methods, we screened for HeV RNA in 183 bat flies collected from flying‐foxes inhabiting a roost in Boonah, Queensland, Australia. It was subsequently demonstrated that during the study period, Pteropus alecto in this roost had a HeV RNA prevalence between 2 and 15% (95% CI [1, 6] to [8, 26], respectively). We found no evidence of HeV in any bat flies tested, including 10 bat flies collected from P. alecto in which we detected HeV RNA. Our negative findings are consistent with previous findings and provide additional evidence that bat flies do not play a primary role in HeV transmission.     

A postal survey of blowfly strike occurrence in two Queensland shires

OBJECTIVE: To describe the occurrence of blowfly strike in sheep flocks in two shires located in south-eastern Queensland. DESIGN: Postal survey of woolgrowers within Stanthorpe and Inglewood shires. PROCEDURE: Questionnaire posted to woolgrowers in June 1999 seeking information on the monthly occurrence of flystrike and number of sheep at risk. RESULTS: Incidence of body strike and breech strike between August 1998 and May 1999 was 1.1 and 0.5%, respectively. The highest incidence of body strike and breech strike was reported in lambs (6.1%) and weaner sheep (1.5%), respectively. The seasonality of the incidence of body strike was bimodal (November and February), whereas the seasonality of the incidence of breech strike was unimodal (April). Occurrence of body strike within a 3-month period in flocks located within 40 to 50 km of each other was significantly (P < 0.05) clustered. CONCLUSION: Body strike appears to be more common than breech strike in the study area, mainly affects lambs and weaners and high-risk periods occur during late spring and autumn. Study results provide valuable information for planning blowfly strike control programs in the area such as timing of management procedures, use of fly traps), and enable the economic impact of flystrike on the industry in this area to be assessed.     

Transmission studies of Hendra virus (equine morbilli-virus) in fruit bats, horses and cats

Objective To determine the infectivity and transmissibility of Hendra virus (HeV). Design A disease transmission study using fruit bats, horses and cats. Procedure Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second exper iment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experi ment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. Results Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated devel oped acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. Conclusions Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus t o horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.     

尼帕病毒G和F蛋白单克隆抗体的制备与鉴定

为制备抗尼帕病毒(NiV)G和F蛋白的单克隆抗体(MAb),本研究以表达NiV G和F蛋白的真核重组表达质粒pCAGG-NiVG和pCAGG-NiVF分别免疫BALB/c小鼠,采用常规技术制备杂交瘤细胞;以表达G和F蛋白的重组牛痘病毒(rWR-NiVG、rWR-NiVF)分别感染BHK细胞,通过间接免疫荧光(IFA)筛...