甲型H1N1流感病毒对未孕雌鼠及孕鼠的致病性研究

目的应用A/Changchun/01/2009(H1N1)(简称XD09)分别感染未孕昆明雌鼠和孕鼠,研究甲型H1N1流感病毒对怀孕哺乳动物的致病性差异。方法 XD09鼻内接种8周龄雌性未孕雌鼠和孕鼠,于感染后第2 d各处死3只,分别检测病毒在鼠脑、鼻甲骨、肺、肝、脾、肾、胎盘(孕鼠)中的分布,同时计算病毒的MLD50,并观察感染鼠临床症状。结果未孕昆明雌鼠感染XD株病毒后第2 d出现被毛粗乱,呼吸窘迫,体重下降;病毒主要分布在肺脏和鼻甲骨,滴度分别为(5.83±0.29)log10eID50/ml和(3.42±0.38)log10eID50/ml,脾组织也检测到病毒的复制;该病毒对未孕雌鼠的MLD50为6.25 log10eID50。昆明孕鼠感染后第3 d开始出现被毛粗乱、活动减少等轻微临床症状,但未见明显的呼吸窘迫,其中1只孕鼠攻毒后流产并产下木乃伊胎儿;病毒主要分布在肺脏和鼻甲骨等呼吸道组织,滴度分别为(5.07±0.55)log10eID50/ml和(4.08±0.29)log10eID50/ml,脑组织也检测到病毒的复制;该病毒对孕鼠的MLD506.5 log10eID50。结论甲型H1N1流感病毒不需要传代适应就能够直接感染未孕雌鼠,而季节性流感病毒通常需要在未孕雌鼠体内连续传代适应后才可在其肺部有效地复制,因此该病毒对未孕雌鼠的致病性要强于季节性流感病毒。研究还发现病毒在未孕雌鼠和孕鼠间表现出不同的组织嗜性,为近一步研究H1N1流感病毒感染孕妇病死率偏高现象建立了动物实验模型。     

荧光定量RT-PCR方法检测感染小鼠不同组织中的H5N1禽流感病毒

目的测定H5N1禽流感病毒感染小鼠各脏器组织中的病毒核酸含量,了解病毒复制与疾病进程的关系,为H5N1禽流感病毒致病机理与防治研究提供技术支持。方法利用建立的荧光定量RT-PCR法对H5N1禽流感病毒感染BALB/c小鼠的不同脏器组织中病毒核酸进行定量检测,观察H5N1禽流感病毒在不同组织中的复制情况。结果利用荧光定量RT-PCR法可从H5N1禽流感病毒感染小鼠后第3d的肺脏、脑、脾脏、肾脏和肝脏组织中检测到不同含量的病毒核酸;而在感染后4d的肺脏、脑和脾脏等组织中的病毒核酸含量开始下降;在感染后第6d,H5N1禽流感病毒核酸在肺脏和脑组织中大量存在外,其余组织中均有少量病毒核酸。结论H5N1流感病毒感染小鼠肺脏和脑组织中病毒核酸含量较高,说明病毒主要在小鼠的肺脏和脑组织中复制。由于荧光定量RT-PCR法可对感染小鼠不同脏器组织中的病毒核酸进行准确定量,可为H5N1禽流感病毒所致疾病的早期诊断、流行病学和致病机理研究提供技术支持。     

荧光定量RT—PCR方法检测感染小鼠不同组织中的HSN1禽流感病毒

目的测定H5N1禽流感病毒感染小鼠各脏器组织中的病毒核酸含量，了解病毒复制与疾病进程的关系，为H5N1禽流感病毒致病机理与防治研究提供技术支持。方法利用建立的荧光定量RT-PCR法对H5N1禽流感病毒感染BALB／c小鼠的不同脏器组织中病毒核酸进行定量检测，观察H5N1禽流感病毒在不同组织中的复制情况。结果利用荧光定量RT-PCR法可从H5N1禽流感病毒感染小鼠后第3d的肺脏、脑、脾脏、肾脏和肝脏组织中检测到不同含量的病毒核酸；而在感染后4d的肺脏、脑和脾脏等组织中的病毒核酸含量开始下降；在感染后第6d，H5N1禽流感病毒核酸在肺脏和脑组织中大量存在外．其余组织中均有少量病毒核酸。结论H5N1流感病毒感染小鼠肺脏和脑组织中病毒核酸含量较高，说明病毒主要在小鼠的肺脏和脑组织中复制。由于荧光定量RT-PCR法可对感染小鼠不同脏器组织中的病毒核酸进行准确定量，可为H5N1禽流感病毒所致疾病的早期诊断、流行病学和致病机理研究提供技术支持。     

河南省濮阳市一起人感染H5N6禽流感流行病学及病原学特征分析

目的 分析2022年3月河南省濮阳市首例人感染H5N6禽流感流行病学特征，探索感染来源，为防控人感染禽流感提供科学依据。方法 对人感染H5N6禽流感病例、密切接触者和涉禽外环境开展流行病学调查，采集病例和密切接触者的呼吸道标本、环境标本、禽类生物样本和禽类产品，运用实时荧光定量PCR方法检测禽流感病毒核酸。结果该病例肺泡灌洗液、咽拭子和鼻拭子样本均检出H5N6亚型禽流感病毒核酸，病例有宰杀禽类职业暴露史。1份屠宰厂的案板涂抹拭子检出H5N6亚型禽流感病毒核酸，9份鸭头类产品的口咽拭子检出H5亚型禽流感病毒核酸，确诊为人感染H5N6禽流感，为单个散发病例。结论 濮阳市首例人感染H5N6禽流感病例为单个散发病例，病原甲型H5N6亚型禽流感病毒可能来自宰杀的鸭子；应加强禽流感日常监测及健康科普。     

鹅源H5N1禽流感病毒在感染雏鸡肾脏的分布及所致病理变化

目的研究鹅源H5N1亚型禽流感毒株人工感染雏鸡后,禽流感病毒在感染雏鸡肾脏内的分布及其所致的病理损伤变化。方法120只1d龄SPF雏鸡随机分为:对照组(C组)、7d龄和21d龄禽流感病毒感染组(Ⅰ、Ⅱ组)。I组和Ⅱ组雏鸡分别于7和21d龄时,分别经鼻、眼、口感染鹅源H5N1亚型禽流感病毒稀释液,3组雏鸡感染后1、3、4、5、7、14、21d,随机抽取5只,心脏采血处死,取肾脏,应用免疫酶组织化学和图像分析技术等手段检测禽流感病毒,并观察肾脏的病理变化。结果雏鸡感染鹅源H5N1亚型禽流感病毒后1～14d,其肾脏血管内皮细胞、肾小管上皮细胞内均可检测到病毒抗原,并呈现不同程度的病理形态损伤。结论鹅源H5N1亚型禽流感病毒对雏鸡肾脏具有较强的嗜组织特性,可造成雏鸡肾脏严重损伤。     

人禽流感死亡病例不同组织器官中H5N1病毒的检测分析

目的了解H5N1病毒在人禽流感死亡病例组织器官中的分布和含量。方法在BSL-3实验室，测定H5N1禽流感病毒分离株的TCID50，并采用荧光定量PCR制作标准曲线；将人禽流感死亡病例的尸解标本，包括心、肝、脾、肺、肾、脑等组织标本研磨处理后，进行荧光定量PCR检测，并根据标准曲线推算H5N1禽流感病毒的病毒载量。结果心、肝、肾、脑组织标本中H5N1禽流感病毒检测为阴性，在肺、脾组织标本中检测到H5N1病毒，病毒载量分别为10^6.3TCID50／ml和10^4.55TCID50／ml。结论除呼吸系统外，在脾组织中也存在H5N1禽流感病毒。     

孕期弓形虫感染对胎鼠体内微量元素的影响

目的探讨刚地弓形虫感染孕鼠后对胎鼠体内微量元素锌（Zn）、铜（Cu）、铁（Fe）含量的影响。方法孕中期BALB/c孕鼠经腹腔接种弓形虫速殖子。分别于孕10、12、14、16和18 d剖杀,取胎盘,制作组织压片及病理切片,观察孕鼠胎盘组织的感染及损伤情况;取胎鼠组织,采用原子吸收分光光谱法测定Zn、Cu、Fe含量,采用DAB增强的Perl＇s铁染色观察胎鼠组织中Fe的分布。结果感染组孕鼠胎盘组织压片可见大量弓形虫速殖子,病理切片HE染色可见炎症细胞浸润及凋亡小体。感染组胎鼠在孕10 d时体内Zn和Fe含量与未感染组比较差异无统计学意义（P〉0.05）,在孕12-18 d均低于未感染对照组（P〈0.05）;Cu含量在孕10、12、14 d与未感染组比较差异无统计学意义（P〉0.05）,在孕161、8 d均低于未感染组（P〈0.05）。感染组胎鼠组织铁染色强度与未感染对照组相比明显减弱。结论孕期感染弓形虫可损伤胎盘组织,进而影响其转运功能,使胚胎体内Zn、Fe和Cu含量降低,影响胚胎的生长发育。     

再测序芯片在北京首例人禽流感病例病原学检测中的应用

目的利用再测序芯片对北京市首例人禽流感病例进行病原筛查和验证。方法采集病例咽拭子和气管抽吸物标本,利用real-ti me RT-PCR进行禽流感病毒H5N1亚型核酸检测;应用病原体再测序芯片对其进行复核,并对其它呼吸道病原体和流感病毒其它亚型进行筛查。结果气管抽吸物标本经real-ti me RT-PCR检测为禽流感病毒H5N1核酸阳性;再测序芯片检测的结果是获得了H5N1的非结构蛋白基因(NS)特异序列,通过与GenBank进行序列比对,确定为禽流感病毒H5N1核酸,并排除了30种流感病毒亚型和其它33种呼吸道病原体的感染。结论病原体再测序芯片具有高灵敏性和特异性,在北京市首例人禽流感病例的病原学筛查和验证中发挥了重要作用。     

重庆市首例人感染H5N6禽流感病例流行病学调查分析

目的 分析重庆市首例人感染H5N6禽流感病例流行病学特征,提高各级医疗机构对“不明原因肺炎”的报告能力,为防制人感染禽流感提供科学依据。方法 于2021-01对重庆首例人感染H5N6禽流感病例、密切接触者、可疑暴露者开展现场流行病学调查,采集生物和环境及食品样本进行实验室检测,运用描述流行病学进行分析。结果 重庆市首例确诊人感染H5N6禽流感病例,咽拭子样本检测结果为H5N6亚型禽流感病毒阳性;34名密切接触者和19名可疑暴露人员未发生H5N6禽流感传播;首例病人有家禽和活禽市场暴露史,日常买菜的活禽市场和活禽市场禽类来源地H5亚型禽流感病毒核酸阳性,活禽市场多处检测出禽流感病毒。结论 重庆市首例人感染H5N6禽流感病例为本地感染,感染来源可能与接触市场活禽有关,非人传人病例。     

孕期弓形虫感染对胎鼠体内微量元素的影响

目的 探讨刚地弓形虫感染孕鼠后对胎鼠体内微量元素锌(Zn)、铜(Cu)、铁(Fe)含量的影响.方法 孕中期BALB/c孕鼠经腹腔接种弓形虫速殖子.分别于孕10、12、14、16和18 d剖杀,取胎盘,制作组织压片及病理切片,观察孕鼠胎盘组织的感染及损伤情况;取胎鼠组织,采用原子吸收分光光谱法测定Zn、Cu、Fe含量,采用DAB增强的Perl's铁染色观察胎鼠组织中Fe的分布.结果 感染组孕鼠胎盘组织压片可见大量弓形虫速殖子,病理切片HE染色可见炎症细胞浸润及凋亡小体.感染组胎鼠在孕10 d时体内Zn和Fe含量与未感染组比较差异无统计学意义(P＞0.05),在孕12～18 d均低于未感染对照组(P＜0.05);Cu含量在孕10、12、14 d与未感染组比较差异无统计学意义(P＞0.05),在孕16、18 d均低于未感染组(P＜0.05).感染组胎鼠组织铁染色强度与未感染对照组相比明显减弱.结论 孕期感染弓形虫可损伤胎盘组织,进而影响其转运功能,使胚胎体内Zn、Fe和Cu含量降低,影响胚胎的生长发育.     

Tropism and Infectivity of Pandemic Influenza A H1N1/09 Virus in the Human Placenta

Influenza virus infection in pregnant women may put the fetus at higher risk; however, to date, there has been no detailed research about the expression of influenza virus receptors in the human placenta. We employed the lectin staining technique, which is a classic influenza virus receptor research method for studying the distribution of viral receptors in the human placenta. In addition, we examined the susceptibility of the human placenta to H1N1/09, by detecting viral proteins and RNA at different time points post-infection. We found that the human placenta expressed both avian and human influenza A virus receptors (α-2, 3-linked sialic acid and α-2, 6-linked sialic acid). In addition, H1N1/09 did not only infect the human placenta, but also replicated and was released into the culture media. We concluded that the human placenta is susceptible to the 2009 influenza A virus (H1N1/09) infection, and that particular attention should be paid to shielding pregnant women from infection during influenza season.     

H6N1亚型禽流感病毒跨种属传播研究进展

禽流感病毒H6N1亚型广泛存在于水禽和陆禽,是最常分离到的甲型流感病毒亚型,遗传分析表明该病毒可能是高致病禽流感病毒H5N1的前体。随着病毒基因的持续进化,H6N1可跨种间屏障传播至哺乳动物,其对于哺乳动物小鼠、猪和雪貂已经具有较强感染能力。血清流行病学调查结果显示少数人H6禽流感病毒抗体阳性,2013年5月,我国台湾出现全球首例人类感染H6N1亚型流感病毒。因此,H6N1病毒宿主范围不断扩大,在这些宿主内可发生病毒基因突变、基因重配,进而演变为具有感染人类潜能的新变异株的可能。本文从病原学、流行病学、病毒感染哺乳动物和人类等方面对H6N1亚型禽流感病毒的研究进展进行综述,以期为H6N1禽流感病毒的防控提供参考。     

H5N1-infected cells in lung with diffuse alveolar damage in exudative phase from a fatal case in Vietnam

Necropsied lung tissues of three fatal cases with avian influenza A virus (H5N1) infection in Vietnam were analyzed to detect H5N1 virus-infected cells. Formalin-fixed and paraffin-embedded lung tissue sections showed typical histological features of diffuse alveolar damage (DAD) in all cases. Immunohistochemistry for the influenza A virus nucleoprotein antigen revealed positive signals of bronchiolar and alveolar epithelial cells in only one patient, who exhibited DAD with an exudative phase and died on the 6th day after onset. However, no signal was detected in the other two cases of DAD with a proliferative phase. These patients died on day 16 and day 17 after onset, respectively. H5N1 virus antigens were detected predominantly in epithelial cells in terminal bronchioles and in alveoli, i.e., type I and type II alveolar pneumocytes, and in alveolar macrophages. The pathogenesis of exudative DAD caused by H5N1 infection is discussed.     

Multiple Neuraminidase Containing Influenza Virus-like Particle Vaccines Protect Mice from Avian and Human Influenza Virus Infection

Avian influenza virus remains a threat for humans, and vaccines preventing both avian and human influenza virus infections are needed. Since virus-like particles (VLPs) expressing single neuraminidase (NA) subtype elicited limited heterosubtypic protection, VLPs expressing multiple NA subtypes would enhance the extent of heterosubtypic immunity. Here, we generated avian influenza VLP vaccines displaying H5 hemagglutinin (HA) antigen with or without avian NA subtypes (N1, N6, N8) in different combinations. BALB/c mice were intramuscularly immunized with the VLPs to evaluate the resulting homologous and heterosubtypic immunity upon challenge infections with the avian and human influenza viruses (A/H5N1, A/H3N2, A/H1N1). VLPs expressing H5 alone conferred homologous protection but not heterosubtypic protection, whereas VLPs co-expressing H5 and NA subtypes elicited both homologous and heterosubtypic protection against human influenza viruses in mice. We observed that VLP induced neuraminidase inhibitory activities (NAI), virus-neutralizing activity, and virus-specific antibody (IgG, IgA) responses were strongly correlated with the number of different NA subtype expressions on the VLPs. VLPs expressing all 3 NA subtypes resulted in the highest protection, indicated by the lowest lung titer, negligible body weight changes, and survival in immunized mice. These results suggest that expressing multiple neuraminidases in avian HA VLPs is a promising approach for developing a universal influenza A vaccine against avian and human influenza virus infections.     

H5N1禽流感病毒抗原基因的体外转录及RNA标准品构建

目的 通过基因克隆和体外转录,获得H5N1禽流感病毒血凝素(HA)、神经氨酸酶(NA)、基质蛋白M(M)基因的RNA片段,为病原学检测提供阳性定量标准品.方法　设计H5N1禽流感病毒的HA、NA及M基因全长开放阅读框的克隆引物,提取H5N1禽流感病毒总RNA,用RT-PCR获得相应片段,分别连接至Pgem-T easy...     

Avian influenza virus infections in humans

Seroepidemiologic and virologic studies since 1889 suggested that human influenza pandemics were caused by H1, H2, and H3 subtypes of influenza A viruses. If not for the 1997 avian A/H5N1 outbreak in Hong Kong of China, subtype H2 is the likely candidate for the next pandemic. However, unlike previous poultry outbreaks of highly pathogenic avian influenza due to H5 that were controlled by depopulation with or without vaccination, the presently circulating A/H5N1 genotype Z virus has since been spreading from Southern China to other parts of the world. Migratory birds and, less likely, bird trafficking are believed to be globalizing the avian influenza A/H5N1 epidemic in poultry. More than 200 human cases of avian influenza virus infection due to A/H5, A/H7, and A/H9 subtypes mainly as a result of poultry-to-human transmission have been reported with a > 50% case fatality rate for A/H5N1 infections. A mutant or reassortant virus capable of efficient human-to-human transmission could trigger another influenza pandemic. The recent isolation of this virus in extrapulmonary sites of human diseases suggests that the high fatality of this infection may be more than just the result of a cytokine storm triggered by the pulmonary disease. The emergence of resistance to adamantanes (amantadine and rimantadine) and recently oseltamivir while H5N1 vaccines are still at the developmental stage of phase I clinical trial are causes for grave concern. Moreover, the to-be pandemic strain may have little cross immunogenicity to the presently tested vaccine strain. The relative importance and usefulness of airborne, droplet, or contact precautions in infection control are still uncertain. Laboratory-acquired avian influenza H7N7 has been reported, and the laboratory strains of human influenza H2N2 could also be the cause of another pandemic. The control of this impending disaster requires more research in addition to national and international preparedness at various levels. The epidemiology, virology, clinical features, laboratory diagnosis, management, and hospital infection control measures are reviewed from a clinical perspective.     

弓形虫感染妊娠中期BALB/c小鼠对胎鼠生长发育及胎盘系数的影响

目的 探讨弓形虫感染妊娠中期BALB/c小鼠对胎鼠生长发育以及胎盘系数的影响.方法在妊娠第8天采用腹腔注射法建立弓形虫感染的动物模型,于妊娠第12、14、16、18天分批处死孕鼠,分离胎盘胎鼠,解剖显微镜下观察胎鼠存活情况,电子天平称量胎盘和胎鼠重量,游标卡尺测量胎鼠体长和尾长. 结果 随着妊娠天数的增加,实验组孕鼠吸收胎明显增多,出现死胎和流产,正常对照组未见异常;正常对照组胎鼠重量、体长和尾长增加明显(胎鼠重量:40.69±11.63,173.43±30.19,495.56±73.97,967.62±83.34:体长:11.53±0.95,16.83±1.15,20.86±1.25;尾长:5.26±0.56,8.08±0.98,10.54±0.58),胎盘重量呈现先增后减趋势(48.62±11.87,87.14±14.05,94.44±12.29,93.33±13.00),胎盘系数呈降低趋势(1.30±0.52,0.51±0.09,0.20±0.05,0.10±0.02),实验组变化趋势不及正常对照组的明显(胎鼠重量:51.11±13.89,148.16 ±36.09,296.11±58.63;体长:11.51±1.42,14.04±1.59;尾长:4.04±0.95,6.87±0.87;胎盘重量:49.31±9.42,66.41±13.28,52.22±12.15;胎盘系数:1.03±0.32,0.48±0.15,0.18±0.05),组内比较差异均有统计学意义(P<0.05);相同时间点的比较显示除第12天组的胎盘胎鼠重量、第14和16天的胎盘系数,还有第14天的胎鼠体长外,其他数据实验组均低于正常对照组,差异有统计学意义(P<0.05). 结论 弓形虫感染妊娠中期BALB/c小鼠可导致胎盘功能障碍,胎鼠生长发育受限,且随着感染天数的增加,可出现死胎和流产等病理性妊娠.     

Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.