流感病毒PR8F的拯救及其对BALB/c小鼠的致病力

为了研究H1N1流感病毒对BALB/c小鼠的致病相关分子机制、传播机制以及开发新型疫苗,将本实验室保存的A/PR/8/34株的8个质粒参照Paulina构建的流感病毒8质粒突变系统做定点突变,利用反向遗传操作技术,成功拯救出H1N1流感病毒突变株PR8F。将PR8F株以106 TCID50/100μL的剂量滴鼻感染BALB/c小鼠,观察小鼠临床表现及体质量变化。解剖攻毒后不同时间小鼠并观察体内主要脏器的病理特征,提取各脏器RNA,通过实时荧光定量PCR方法,检测PR8F株在小鼠体内不同脏器中的病毒残留量和在BALB/c小鼠肺脏中的扩增效率。结果表明,H1N1流感病毒突变株PR8F感染小鼠4~7d后全部致死;PR8F株在小鼠体内各脏器中的病毒残留量有很大差异,肺脏中最多,且病毒在肺脏内呈指数形式扩增。本试验建立了H1N1流感病毒PR8F株对BALB/c小鼠的感染模型,为H1N1流感病毒致病机制和传播机制的研究奠定基础。     

广西部分地区猪链球菌的分离鉴定及小鼠致病性研究

对来自广西边境地区部分发病猪场及屠宰场的370份猪组织进行细菌分离鉴定,共分离到16株链球菌,对这16株链球菌进行了形态学观察、PCR鉴定及小鼠致病性试验。结果表明,其形态、染色、生化特性均符合猪链球菌的特点,通过GDH鉴定与测序,证实这16株菌株均为猪链球菌,分别命名为GX1~GX16。通过特异性引物PCR分型（35个血清型）,其中有1株为1型,1株为2型,1株为5型,1株为29型,5株为8型,7株未分出型。用这16株猪链球菌对昆明小鼠和BALB/c小鼠进行动物致病性试验,结果发现,菌株GX10对昆明小鼠和BALB/c小鼠致死率均为100%;菌株GX11对BALB/c小鼠致死率为80%;其他菌株对昆明小鼠和BALB/c小鼠均无致病性。菌株GX10、GX11对BALB/c小鼠的LD₅₀分别为4.5×10⁵和3.6×10⁹ CFU。本试验结果表明,广西边境地区存在致病性猪链球菌8型。BALB/c小鼠对猪链球菌的敏感性高于昆明小鼠,更适合作为猪链球菌致病性研究的动物模型。     

H5N1亚型猪流感病毒A／Swine／Fujian／1／01感染性克隆构建及其对小鼠致病性研究

2001年从福建某猪场分离到1株H5N1亚型猪流感病毒(SIV)A/Swine/Fujian/1/01(SW/FJ/1/01).SW/FJ/1/01对小鼠具有强致病性,致死率为100%,为进一步研究SW/FJ/1/01对BALB/c小鼠致病性的分子基础,本研究构建了SW/FJ/1/01的8个节段重组质粒构成的反向基因操作系统,成功拯救了病毒(R-SW/FJ/1/01).R-SW/FJ/1/01和野生型SW/FJ/1/01对BALB/c小鼠致病性没有差别.SW/FJ/1/01反向基因操作系统的建立为进一步阐明H5N1亚型SIV对哺乳动物模型BALB/c小鼠的致病机制等方面的研究奠定了基础.     

A型流感病毒致病性及跨种间传播的分子生物学基础

A型流感病毒在温血动物中广泛存在,也是目前导致人类和各种动物流感疾病的主型。在自然情况下流感病毒感染的宿主范围有一定的特异性,分离自人的流感病毒一般不能在鸡、鸭等禽类体内复制,同样禽流感病毒在灵长类动物体内的复制能力也极差。但近年来不断发生禽流感病毒（包括H5N1,H9N2,H7N7亚型病毒）直接传染给人的事件,而2009年爆发的人类的H1N1流感的病原则是猪源流感病毒重组而来。研究证实,流感病毒的致病性及跨种间传播的深层原因是病毒分子结构差异及其与宿主细胞相互作用。病毒多种结构蛋白和非结构蛋白某些功能位点上氨基酸的差异是病毒致病性及其跨中传播的分子生物学基础。     

H6N6亚型禽流感病毒反向遗传操作系统的建立

为建立H6N6亚型禽流感病-毒(AIV) A/Chicken/GuangDong/S1311/10 (W-CKGD)反向遗传操作系统,本研究构建了W-CKGD的8个重组质粒,拯救出AIV A/Chicken/GuangDong/S1311/10 (R-CKGD).全基因序列测定结果表明,救获病毒R-CKGD与野生病毒W-CKGD的核苷酸序列完全相同.R-CKGD与W-CKGD具有相同的受体结合特性和对BALB/c小鼠均呈低致病性,以106 EID50剂量鼻腔感染BALB/c小鼠,病毒仅在小鼠的呼吸道复制,可以引起小鼠体重下降但均不造成死亡.实验结果表明,R-CKGD与W-CKGD保持了一致的生物学特性,从而为进一步研究H6亚型AIV的致病机理及跨宿主传播机制等提供了良好的平台.     

1株H5N1禽流感病毒云南分离株的生物学特性分析

为了解2014年分离自云南省通海县的1株H5N1亚型禽流感病毒的生物学特性,对病原A/chicken/Yunnan/1/2014(yn2014)进行了一系列分析,包括全基因组序列测定,遗传演化分析,抗原性分析,以及对SPF鸡、鸭和BALB/c小鼠的致病性试验。交叉血凝抑制试验和免疫保护试验对yn2014分离株进行抗原分析显示,yn2014与Re-5疫苗株之间抗原性差异显著。全基因组序列测定和遗传进化分析表明,yn2014病毒HA基因与韩国、日本、中国流行毒株核苷酸高度同源,同属于2.3.4.6分支;HA含有多个连续碱性氨基酸,具有高致病性禽流感病毒的分子特征。yn2014病毒人工静脉接种SPF鸡和鼻腔感染SPF鸭试验,结果显示,yn2014病毒对鸡、鸭的致死率分别为100%和50%。用10~6EID50病毒鼻腔感染6周龄BALB/c小鼠,结果显示,yn2014病毒对BALB/c小鼠的致病性较低,仅在鼻窦中复制,且不能在小鼠之间传播。本研究为该分支禽流感病毒的防控提供了较好的参考价值。     

人工感染牛无浆体BALB/c小鼠内脏器官的组织病理学观察

为了解牛无浆体阳性动物内脏器官的病理变化,将牛无浆体人工接种至BALB/c小鼠,利用血涂片染色法和PCR法鉴定感染情况,对感染牛无浆体的BALB/c小鼠内脏器官做组织病理学观察。结果表明,牛无浆体可引起BALB/c小鼠心脏的心肌细胞嗜酸性增强、胞核固缩,肝脏的肝细胞中重度颗粒变性,脾脏的淋巴细胞形态不规则等病变。本研究表明,感染牛无浆体的BALB/c小鼠的内脏器官和心、肝、脾等均产生了不同程度的病理学变化,为牛无浆体的致病性研究提供了参考数据。     

甲流重组犬流感病毒的小鼠致病性研究

本研究通过建立犬流感病毒(Canine influenza virus,CIV)感染BALB/c小鼠模型,探索新发现的甲流重组CIVs对小鼠的致病性。我们采用9株H1N1和1株H3N2甲流重组CIVs,以106 TCID_(50)/mL的剂量滴鼻攻毒10组小鼠。结果表明:感染后4 d小鼠体重均有一定程度的下降,其中A/Canine/Guangxi/LZ56/2015(H1N1)(简称LZ56)组的小鼠体重下降最为明显(P0.5),其存活率为77.78%;通过TCID_(50)测定发现10株甲流重组CIVs均能在小鼠的鼻窦和肺脏复制,以LZ56组的复制滴度最高,分别达到107.3 TCID_(50)/mL和10~(5.3) TCID_(50)/mL;肺脏病理切片观察发现LZ36、LZ45、LZ56和QZ5组出现不同程度病变,以支气管上皮细胞坏死脱落,肺泡壁增厚,肺泡腔以及支气管内有大量的弥漫性浸润的单核细胞为主要特征。结果表明,新型重组CIVs均能感染BALB/c小鼠,并能在小鼠体内较好复制,甚至致死,一旦这些新型CIVs以犬为中间宿主传播给人,势必对人类健康构成重大威胁。因此,加强对CIVs的监测将为防控人流感大暴发提供提前预警。     

A型流感病毒聚合酶复制必需的宿主因子及其作用机制

正A型流感病毒是引起人和动物流感的主要病原,对人类的生命健康和社会生活形成巨大威胁。A型流感病毒亚类较多,感染宿主较广,且可以跨种传播进而造成流感大流行,因此A型流感病毒的跨种传播机制一直是本领域的研究热点。流感病毒的RNA聚合酶是该病毒在宿主细胞内转录与复制的物质基础,在病毒的跨物种传播中发挥重要作用。其需借助某些宿主蛋白来完成病毒的转录和复制过程,目前已经发现有多种宿主因子     

一株pH1N1/2009猪流感病毒的进化分析与生物学特性研究

为了解SIV A/swine/Liaoning/5/2014(H1N1)分离株的生物学特性与遗传进化关系,本研究对该分离株的全基因序列进行测定,并对其进行小鼠致病性实验。基因组序列分析表明:该病毒的8个基因节段均来自A型H1N1流感病毒。HA蛋白裂解位点为~(325)PSIQSRG~(331),属于低致病性流感病毒的分子特征,其HA基因与A/Xia Men/SWL1280/2013(H1N1)的相似性达到98.9%。对动物致病性试验结果显示:该病毒可以感染哺乳动物模型BALB/c小鼠,但仅能够在鼠的肺脏和鼻甲骨粘膜上皮细胞中复制,病毒滴度分别为5.5 log_(10)EID_(50)/m L和3.3 log_(10)EID_(50)/mL,该病毒株对小鼠的生长具有抑制作用,可以导致感染后7 d内小鼠体质量下降15.2%,随后逐渐恢复,但始终低于正常对照小鼠,呈现一定的致病性。本研究为该病毒已在我国动物群体内流行提供了新的证据。     

Review of Influenza A Virus in Swine Worldwide: A Call for Increased Surveillance and Research

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high‐priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.     

The Inability to Screen Exhibition Swine for Influenza A Virus Using Body Temperature

Agricultural fairs create an unconventional animal–human interface that has been associated with swine‐to‐human transmission of influenza A virus (IAV) in recent years. Early detection of IAV‐infected pigs at agricultural fairs would allow veterinarians to better protect swine and human health during these swine exhibitions. This study assessed the use of swine body temperature measurement, recorded by infrared and rectal thermometers, as a practical method to detect IAV‐infected swine at agricultural fairs. In our first objective, infrared thermometers were used to record the body surface temperature of 1,092 pigs at the time of IAV nasal swab collection at the end of the exhibition period of 55 agricultural fairs. IAV was recovered from 212 (19.4%) pigs, and the difference in mean infrared body temperature measurement of IAV‐positive and IAV‐negative pigs was 0.83°C. In a second objective, snout wipes were collected from 1,948 pigs immediately prior to the unloading of the animals at a single large swine exhibition. Concurrent to the snout wipe collection, owners took the rectal temperatures of his/her pigs. In this case, 47 (2.4%) pigs tested positive for IAV before they entered the swine barn. The mean rectal temperatures differed by only 0.19°C between IAV‐positive and IAV‐negative pigs. The low prevalence of IAV among the pigs upon entry to the fair in the second objective provides evidence that limiting intraspecies spread of IAV during the fairs will likely have significant impacts on the zoonotic transmission. However, in both objectives, the high degree of similarity in the body temperature measurements between the IAV‐positive and IAV‐negative pigs made it impossible to set a diagnostically meaningful cut point to differentiate IAV status of the individual animals. Unfortunately, body temperature measurement cannot be used to accurately screen exhibition swine for IAV.     

Perceptions and attitudes of swine exhibitors towards recommendations for reducing zoonotic transmission of influenza A viruses

Since 2011, there have been 468 cases of variant influenza A virus (IAV) reported in the United States, many of which were associated with youth swine exhibition. In an effort to mitigate risk associated with exposure to IAV in swine, the “Measures to Minimize Influenza Transmission at Swine Exhibitions” (MtM) was developed for show organizers, volunteers and exhibitors. These recommendations are updated annually; however, it is not clear if youth swine exhibitors are aware of the recommendations; support the recommendations; and would be willing to practise recommended behaviours. Therefore, a cross‐sectional survey method was used to assess swine exhibitor perceptions and their adoption of swine production practices aimed at reducing the transmission of IAV at the human–animal interface. In addition, the survey asked participants their state of residence and the number of shows they would attend in 2017. In all, 155 participants who showed swine on a regular basis (x? = 11 shows per year), from at least 18 states within the US, completed the survey. At least, 67% of participants believed each statement was a good recommendation, with 6 of 11 recommendations being supported by >90% of participants. When asked if recommendations could be implemented, 65%–94% of respondents agreed, and 21%–89% of participants had already implemented each recommendation, respectively. Although significant efforts have been made to increase signage at swine exhibitions (warning of risks associated with eating/drinking in animal areas), a majority of respondents report eating/drinking in the barn and are unwilling to change their behaviours. This study provides evidence that developing and disseminating static recommendations to reduce zoonotic disease transmission is not enough to change human behaviour to prevent future variant IAV infections associated with swine exhibitions.     

小鼠细菌性乳腺炎模型研究进展

奶牛细菌性乳腺炎一直是困扰奶牛养殖业的一大难题,给奶牛养殖行业造成巨大经济损失,直接危害奶牛健康。为有效治疗奶牛细菌性乳腺炎,研究人员需要通过动物模型来进行相关研究,以期找到优良的治疗方法。随着动物模型多年的探索与使用,小鼠细菌性乳腺炎模型因具有省时、省力以及性价比高的优点获得了研究人员的广泛认可,被大量用于相关研究中。就此对小鼠细菌性乳腺炎模型的建立、特点以及应用进行综述,旨在为细菌性乳腺炎相关研究提供参考。     

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.     

Serological surveillance and factors associated with influenza A virus in backyard pigs in Southern Brazil

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP‐ELISA, subtype‐specific serology based on hemagglutination inhibition (HI) assay of the ELISA‐positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%–7.33%) and 2.3% (1.34%–3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as “age,” “sex,” “number of suckling pigs” and “neighbours raising pigs” that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human‐to‐pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.     

甲型流感病毒与NLRP3炎性小体激活的研究进展

甲型流感病毒(influenza A virus, IAV)对全人类的健康构成了持续性威胁,感染时会引发炎症性疾病。NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎性小体是一种天然免疫系统传感器,与甲型流感病毒感染引发的炎症反应相关。在宿主细胞被甲型流感病毒感染时,能够激活NLRP3炎性小体,促使pro-IL-1β和pro-IL-18剪切成熟的IL-1β和IL-18并分泌至胞外,进而引发炎症反应。研究表明,NLRP3炎性小体对于在甲型流感病毒感染期间诱导先天性免疫应答至关重要,并且由甲型流感病毒感染引发过度激活的NLRP3炎性小体与细胞因子风暴和不受控制的炎症反应有关。本文回顾了甲型流感病毒与NLRP3炎性小体之间关系的研究进展,并讨论了NLRP3炎性小体在甲型流感病毒致病机理中的保护作用和致病作用。     

甲型流感病毒NS1蛋白研究进展

甲型流感病毒(Influenza A virus,IAV)是引起每年季节性流感及偶尔的流感大流行的主要病原体,严重危害公共卫生和社会经济.IAV属于分节段的单股负链RNA病毒,其基因组编码多种不同的蛋白质,其中非结构蛋白1(non-structural protein 1,NS1)是IAV的调节宿主免疫应答和病毒致病性...     

Paradigm shifts in vaccine development: lessons learned about antigenicity,pathogenicity and virulence of Brucellae

As part of a program to support the USDA Animal Plant Health Inspection Service Bovine Brucellosis Eradication Program, the Brucellosis Research Unit of the National Animal Disease Center (NADC) sought to develop a bovine brucellosis vaccine that would allow vaccinated animals to be distinguished from virulent field infected animals. In order to meet that goal, several avenues of research were undertaken to construct and test candidate vaccines, including Brucella abortus RB51. In early vaccine development studies, a subunit preparation obtained by extracting B. abortus with salts was studied as a candidate subunit vaccine. Later, molecular biological techniques were used both to clone genes encoding products found in the salt extract (BCSP31 and Cu–Zn SOD) and genes encoding proteins of B. abortus that were antigenic (HtrA) or possibly essential (two-component systems) for full virulence of B. abortus. In vitro systems using mammalian cells lines such as HeLa and macrophage-related were used along with the mouse model and host animal models. Results obtained at NADC and in other Brucellosis research laboratories, using survival in mammalian cell lines and the mouse model to access pathogenicity and virulence of genetically engineered strains, do not necessarily identify loci that are essential for full virulence or pathogenicity in the natural host, the bovine. Studies at NADC and other brucellosis laboratories showed that antigenicity was not a predictor of the effectiveness of a protein as a subunit vaccine.     

Genetic analysis of human and swine influenza A viruses isolated in Northern Italy during 2010–2015

Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants—possibly endowed with pandemic potential—and emphasize the importance of continuous surveillance at both animal and human level.