痘病毒科不同病毒属特异的PCR检测方法的建立

目的:建立可准确、快速地鉴别诊断可感染人的不同属痘病毒的特异PCR方法。方法:设计针对正痘病毒属、副痘病毒属和传染性软疣病毒属的多对特异引物,并制备相应的DNA模板,针对不同的模板优化引物与反应条件,分别进行检测筛选,建立病毒属特异的单独与多重PCR方法。结果:单一模板的PCR扩增反应中,正痘病毒的检测敏感性可达101拷贝/μL(引物为OPEaL-F1880/OPEaL-R2057),副痘病毒的检测敏感性可达101拷贝/μL(引物为PP2/PP3),传染性软疣病毒的检测敏感性为100 pg/μL体系(引物为MCV1/MCV2);混合模板的PCR扩增反应中,各属特异的引物均可获得预期大小的特异片段。结论:我们建立的PCR诊断方法,可用于痘病毒科不同病毒属感染的实验室特异快速鉴别诊断。     

猴痘的流行现状及防控

猴痘(monkeypox)是由猴痘病毒感染所致的人兽共患病,主要发生在非洲中部、西部地区。猴痘病毒可感染多种哺乳类动物,主要在动物中流行,人接触感染动物后可被传染。猴痘的临床表现与天花相似(发热、皮疹等),但症状较轻。天花疫苗接种可提供预防猴痘的免疫保护力。然而,因全球天花被消灭而停止接种天花疫苗后,猴痘成为最可能威胁人类的正痘病毒性疾病。近期,其散发病例在欧洲多地出现。2022年5月7日英国报道了猴痘疫情。随后,欧洲报道猴痘确诊和疑似病例超过100例。猴痘主要传播途径包括接触感染动物、与患者直接接触或间接接触。2022年5月20日,世界卫生组织就此次猴痘疫情召开了紧急会议,旨在提高对猴痘的认识,做好防范应对准备。世界卫生组织、美国疾病预防控制中心、英国卫生部门报告了相关疫情并制定了相应的防控措施。截至2022年5月28日我国尚无输入性猴痘报道,但因国际交往频繁等仍须提高警惕。本文介绍了猴痘流行现状及有关防控信息,以供借鉴。     

李痘病毒实时荧光定量RT-PCR检测方法的建立

目的:建立李痘病毒(PPV)特异、灵敏、快速的实时荧光定量RT-PCR检测方法,用于核果类种苗的健康评测及李痘病毒疫情监测。方法:根据PPV-D株系和PPV-M株系的外被蛋白(CP)基因保守序列,设计特异性引物和TaqMan探针,扩增全长CP基因片段,并将其克隆到pMD18-T载体上,构建质粒标准品,建立PPV的实时荧光定量RT-PCR检测方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:此荧光定量RT-PCR方法对PPV检测呈现高灵敏度和高特异性,与马铃薯Y病毒和马铃薯X病毒无交叉反应,最低检出限可达1.6×102拷贝/μL,标准曲线的相关系数为0.999 18。结论:建立了李痘病毒的荧光定量RT-PCR检测方法,可望应用于检验检疫部门对李痘病毒的快速检测。     

猴痘病毒的分类、演化和宿主范围:痘病毒的系统综述和分析（英文）

近20年来,偶发的感染人类的猴痘疾病严重影响了公共卫生和经济。啮齿类动物是猴痘病毒（MPXV）在自然界的主要宿主。MPXV可以通过直接接触传播至人并引起自限性感染,出现皮疹和淋巴结症状。本世纪初,美国报道了第一例在非洲以外地区人感染MPXV的病例,此后,猴痘逐步在全世界流行,并成为严重的公共威胁。随着病毒检测和测序技术的发展,许多新的痘病毒被发现,从而丰富了痘病毒科的多样性。本综述对MPXV以及痘病毒科中的其他病毒的最新分类、宿主范围、基因组结构特点、致病性以及遗传进化关系进行了全面概述。这些信息将促进我们对痘病毒的遗传进化关系和传播的了解,并且对未来病毒的研究和疾病的控制提供帮助。     

检测EB病毒TK激酶抗体方法的建立及其在鼻咽癌诊断中的应用

目的:建立检测鼻咽癌(NPC)患者血清中EB病毒TK激酶的酶免疫测定(EIA)方法,用于筛选和辅助诊断早期NPC患者。方法:用基因工程高效表达的EB病毒TK激酶作为包被抗原,建立EIA检测方法,检测NPC患者和对照血清中的TK/IgG抗体。结果:在NPC患者血清中检出了特异的对EB病毒TK激酶的IgG抗体,而正常人血清中为抗体阴性。结论:首先建立了简便、快速、特异和敏感的早期诊断NPC的EIA方法,并作为我公司产品扩展应用。     

重组痘病毒表达SARS冠状病毒纤突蛋白及其免疫原性分析

对牛痘病毒弱毒株表达的SARS冠状病毒纤突蛋白(Spike protein,S)的免疫原性进行分析与比较.以减毒痘病毒(WR株)为载体重组了SARS冠状病毒全长S基因(rWR-SARS-S).SDS-PAGE和Western blot试验表明,迁移率约为190kD的重组SARS S蛋白可在HeLa细胞中表达,而且可以被鸡抗SARS全病毒高免血清识别,具有特异免疫反应原性.进一步研究表明,rWR-SARS-S感染的细胞在IFA试验中可与鸡抗SARS的高免血清发生特异反应,具有良好的敏感性和特异性.以104PFU的rWR-SARS-S免疫BALB/c小鼠产生的抗体在间接ELISA试验中可以被S蛋白识别,产生特异抗原抗体反应.利用痘病毒表达的SARS冠状病毒S蛋白具有良好的抗原性和生物学活性,可替代SARS冠状病毒全病毒,为研究安全、敏感和特异的重组诊断抗原奠定了重要基础.     

实时荧光PCR检测巴西孢子丝菌的实验方法的建立

目的建立一种快速、特异、灵敏的荧光PCR方法检测巴西孢子丝菌。方法比对NCBI数据库中所有巴西孢子丝菌内转录间隔区(internal transcribed spacer, ITS)序列,在保守区域设计并合成特异性引物和探针,建立并优化荧光PCR检测方法。对优化后的方法使用标准浓度核酸进行扩增效率、灵敏度及特异度评价。通过巴西孢子丝菌小鼠感染模型,与组织培养比较,对本研究中的方法进行评价。结果建立的实时荧光PCR方法对巴西孢子丝菌的检测限为100fg。该方法对申克孢子丝菌、球形孢子丝菌、其他常见致病真菌28种、常见细菌3种以及人类基因组和小鼠基因组扩增结果均为阴性,特异度为100%。对巴西孢子丝菌感染小鼠脑、肝、肺、脾、肾及淋巴结检测与培养结果相一致。结论本研究建立的荧光PCR方法可快速、灵敏、特异地鉴定巴西孢子丝菌,并能够有效的对感染小鼠模型标本进行检测,有助于孢子丝菌病的早期特异性病原学诊断。     

表达HIV-1 gag重组鸡痘病毒的构建与筛选

构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。     

表达猴免疫缺陷病毒Env蛋白的重组鸡痘病毒的构建和筛选

目的:构建并筛选表达猴免疫缺陷病毒(SIV)Env蛋白的重组鸡痘病毒(FPV),并对其进行鉴定。方法:设计引物,通过PCR技术扩增SIV env基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的FPV穿梭载体pTKET中,获得重组质粒pTKET-SIV env,然后将其与FPV282E4株共转染原代鸡胚成纤维细胞进行同源重组,以增强型绿色荧光蛋白为筛选标记,通过噬斑筛选获得重组病毒,应用PCR、RT-PCR、Western印迹对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组FPV基因组中,RT-PCR、Western印迹结果表明SIV Env蛋白在感染细胞内表达且具有抗原性;连续传代20次,PCR、RT-PCR、Western印迹均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,且病毒已经纯化。结论:获得表达SIV Env蛋白的重组FPV,为进一步免疫试验研究奠定了基础。     

痘病毒非结构蛋白拮抗IFN-I介导免疫逃逸机制研究进展

近些年,猴痘病毒、牛结节性皮肤病病毒等痘病毒给公共卫生和畜牧业健康发展带来了巨大危害,引起全球广泛关注。痘病毒（Poxviridae）是一种大型双链DNA病毒,宿主范围广。在与宿主长期作用过程中,痘病毒通过编码非结构蛋白来逃逸宿主IFN-I介导的免疫反应。本文综述了痘病毒编码的非结构蛋白拮抗IFN-I天然免疫反应的研究进展,为防控痘病毒传播提供理论依据,为痘病毒新型疫苗研发及药物靶点筛选提供参考。     

LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.     

Monkeypox virus: a re-emergent threat to humans

Human monkeypox (MPX) is a rare zoonotic infection characterized by smallpox-like signs and symptoms. It is caused by monkeypox virus (MPXV), a double stranded DNA virus belonging to the genus Orthopoxvirus. MPX was first identified in 1970 and mostly prevailed in the rural rainforests of Central and West Africa in the past. Outside Africa, MPX was reported in the United Kingdom, the USA, Israel, and Singapore. In 2022, the resurgence of MPX in Europe and elsewhere posed a potential threat to humans. MPXV was transmitted by the animals-human or human-human pathway, and the symptoms of MPXV infection are similar to that of smallpox, but in a milder form and with lower mortality (1%–10%). Although the smallpox vaccination has been shown to provide 85% protection against MPXV infection, and two anti-smallpox virus drugs have been approved to treat MPXV, there are still no specific vaccines and drugs against MPXV infection. Therefore it is urgent to take active measures including the adoption of novel anti-MPXV strategies to control the spread of MPXV and prevent MPX epidemic. In this review, we summarize the biological features, epidemiology, pathogenicity, laboratory diagnosis, and prevention and treatment strategies on MPXV. This review provides the basic knowledge for prevention and control of future outbreaks of this emerging infection.     

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.     

Human monkeypox

Human monkeypox, occurring in the tropical rainforest of west and central Africa, is regarded as the most important orthopoxvirus infection for epidemiological surveillance during the post-smallpox era. This disease, first recognized in Za?re in 1970 resembles smallpox clinically but differs epidemiologically. Clinical features, their evolution and sequelae of monkeypox could be compared with discrete ordinary or modified type of smallpox. A case-fatality rate of 14% has been observed but some cases can be exceedingly mild or atypical and may easily remain undetected and unreported. Pronounced lymphadenopathy has been the only clinical feature found commonly in monkeypox but not in smallpox. Fifty-seven cases of human monkeypox have occurred since 1970, in the tropical rainforests in six west and central African countries, the majority of them (45) being reported from Za?re. The disease appears to be more frequent in dry season. Children below ten years of age comprise 84% of the cases. Smallpox vaccination protects against monkeypox. Clusters of cases have been observed in certain areas within countries and within affected households. Human-to-human spread has possibly occurred seven times. No cases of possible tertiary spread were observed. The secondary attack rate among susceptible close household contacts was 10%, among all susceptible contacts 5%. This is much lower than that occurring with smallpox, which is between 25-40%. The limited avidity of monkeypox virus for human beings indicates that monkeypox is probably a zoonosis, although the animal reservoir(s) have not yet been identified. The low transmissibility, resulting in low frequency of disease in man indicates that monkeypox is not a public health problem. Human monkeypox has been a relatively newly recognized disease. Studies are in progress to identify the natural cycle of monkeypox virus and to define better its clinical and epidemiological characteristics. Special surveillance is maintained in endemic areas with the aim to provide assurance that in spite of waning immunity of the human population following cessation of the smallpox vaccination, the disease does not constitute a potential danger to man.     

Tenth anniversary of smallpox eradication: Results of epidemiological and virological surveillance and studies in the post-eradication period

In accordance with recommendations of the Global Commission on the certification of smallpox eradication for the 10-year period after the eradication of this infection, all suspected cases of smallpox have been thoroughly checked up, and in none of them the diagnosis of smallpox has been confirmed. The study of monkeypox in humans has revealed that this zoonosis is spread over a wider area than supposed earlier and covers 7 countries of Equatorial Africa, occurring most frequently in Zaire. In about 70% of cases of monkeypox in human the disease is contracted from animals serving as natural virus carriers and in about one-third of such cases, from humans having monkeypox. The infectivity of humans with monkeypox for persons having close contacts with them is somewhat lower (12.3%) than in smallpox when this characteristic varies from 37% to 88%. Monkeypox in humans may take an asymptomatic course. Some species of tropical squirrels serve as natural virus carriers. These investigations have also resulted in essential corrections being made in understanding the ecology of cowpox virus, another orthopoxvirus pathogenic for man. At least 5 species of rodents have proved to be of interest as natural carriers of cowpox virus.     

Effective antiviral treatment of systemic orthopoxvirus disease: ST-246 treatment of prairie dogs infected with monkeypox virus

Smallpox preparedness research has led to development of antiviral therapies for treatment of serious orthopoxvirus infections. Monkeypox virus is an emerging, zoonotic orthopoxvirus which can cause severe and transmissible disease in humans, generating concerns for public health. Monkeypox virus infection results in a systemic, febrile-rash illness closely resembling smallpox. Currently, there are no small-molecule antiviral therapeutics approved to treat orthopoxvirus infections of humans. The prairie dog, using monkeypox virus as a challenge virus, has provided a valuable nonhuman animal model in which monkeypox virus infection closely resembles human systemic orthopoxvirus illness. Here, we assess the efficacy of the antiorthopoxvirus compound ST-246 in prairie dogs against a monkeypox virus challenge of 65 times the 50% lethal dose (LD(50)). Animals were infected intranasally and administered ST-246 for 14 days, beginning on days 0, 3, or after rash onset. Swab and blood samples were collected every 2 days and analyzed for presence of viral DNA by real-time PCR and for viable virus by tissue culture. Seventy-five percent of infected animals that received vehicle alone succumbed to infection. One hundred percent of animals that received ST-246 survived challenge, and animals that received treatment before symptom onset remained largely asymptomatic. Viable virus and viral DNA were undetected or at greatly reduced levels in animals that began treatment on 0 or 3 days postinfection, compared to control animals or animals treated post-rash onset. Animals treated after rash onset manifested illness, but all recovered. Our results indicate that ST-246 can be used therapeutically, following onset of rash illness, to treat systemic orthopoxvirus infections.     

Independent evolution of monkeypox and variola viruses.

Smallpox was eradicated more than 10 years ago, but infection with another Orthopoxvirus, monkeypox virus, can result in a clinical picture resembling smallpox. Human infection with monkeypox virus is extremely rare, not easily transmitted, and confined to the rain forest belt of Africa (Z. Jezek and F. Fenner, p. 81-102, in Human Monkeypox, 1988). Evidence that variola virus, the causative agent of smallpox, might be readily derived from monkeypox virus was presented [S. S. Marennikova and E. M. Shelukhina, Nature (London) 276:291-292, 1978; S. S. Marennikova, E. M. Shelukhina, N. N. Maltseva, and G. R. Matsevich Intervirology 11:333-340, 1979], but this was not confirmed [K. R. Dumbell and L. C. Archard, Nature (London) 286:29-32, 1980] and was subsequently discounted (J. J. Esposito, J. H. Nakano, and J. F. Obijeski, Bull. W.H.O. 63:695-703, 1985). Although enough difference between the genomes of monkeypox and variola viruses to rule out a simple interconversion has been demonstrated [K. R. Dumbell and L. C. Archard, Nature (London) 286:29-32, 1980; J. J. Esposito and J. C. Knight, Virology 143:230-251, 1985; J. J. Esposito, J. H. Nakano, and J. F. Obijeski, Bull. W.H.O. 63:695-703, 1985; M. Mackett and L. C. Archard, J. Gen. Virol. 45:683-701, 1979], the possibility that monkeypox virus was a more remote ancestor of variola virus remained. We have now identified a sequence in monkeypox virus DNA which is a homolog of a 1,065-bp open reading frame in the conserved region of the variola virus genome but which has multiple deletions. This is strong evidence that monkeypox virus is not ancestral to variola virus and strengthens confidence in the long-term success of smallpox eradication.     

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).