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。     

H5N1禽流感病毒感染孕鼠的研究

目的用H5N1禽流感病毒感染昆明孕鼠,检测病毒在感染孕鼠各组织脏器中的复制及分布情况,并证明病毒能否通过孕鼠的胎盘垂直传染给胎鼠。方法用虎源H5N1亚型禽流感病毒滴鼻感染妊娠10-12 d的昆明孕鼠,观察孕鼠感染后的临床症状。接种病毒后第3、4、5、6和7 d分别处死3只孕鼠,取孕鼠的肺、脑、脾、肾、子宫、胎盘及胎鼠,利用RT-PCR、Real-time PCR和病毒分离方法检测各组织中的病毒核酸和病毒滴度,并进行病理组织学与免疫组织化学检测。结果昆明孕鼠接种病毒后第3 d,即可在肺、脑、脾、肾、子宫及胎盘组织中检测出H5N1禽流感病毒核酸,并从子宫、胎盘分离出H5N1禽流感病毒;感染后第6 d,从胎鼠体内检测到病毒核酸并分离出H5N1禽流感病毒。结论H5N1亚型禽流感病毒可以感染孕鼠,在孕鼠子宫和胎盘复制,感染后期可通过胎盘屏障传给胎鼠。     

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。     

禽流感病毒感染小鼠模型病理损伤及抗原定位研究

目的通过建立高致病性禽流感H5N1病毒感染小鼠模型,研究病毒感染致小鼠组织病理损伤、病毒抗原在体内分布及复制特点,为禽流感病毒传播途径及致病机理研究提供实验依据。方法采用A/vietnam/1194/2004(H5N1)病毒株,滴鼻感染BALB/c小鼠,5 d后取小鼠肺、脑、脾、肠、心、肝、肾等组织,制备石蜡切片并进行组织病理损伤和抗原定位研究。结果小鼠肺组织表现为弥漫性肺损伤,脑组织出现轻度炎症反应,脾组织结构完整但脾小体可见凋亡细胞,其它脏器组织结构未查见组织病理损伤;利用M2、NS1单抗,采用免疫组织化学方法检测发现小鼠肺、脑、肠等均有M2、NS1抗原分布。结论高致病性H5N1病毒感染可在小鼠肺、脑、肠等多种组织中复制,引起严重的肺组织病理损伤及轻度的脑组织及脾组织病理损伤。     

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。     

成人重症肺炎患者EB病毒和人鼻病毒混合感染的病原学分子鉴定

目的对1例有禽类接触史的成人重症肺炎患者进行病原学研究,确定其病原体。方法采集患者的下呼吸道标本,应用荧光PCR方法检测禽流感H5N1和SARS病毒及甲型H1N1和季节性流感病毒;应用多重PCR方法检测12种常见呼吸道病毒;应用普通PCR方法检测EB病毒和巨细胞病毒,扩增阳性的病毒基因片段测序后,与GenBank数据库参考序列进行同源性分析。结果荧光PCR法检测禽流感病毒H5N1病毒和SARS病毒核酸阴性,甲型H1N1和季节性流感病毒核酸阴性;多重PCR检测人鼻病毒、EB病毒均阳性;普通PCR检测EB病毒核酸阳性。基因序列比对显示,EB病毒核苷酸序列与GenBank数据库中参考序列同源性为99.5%～100%;人鼻病毒的核苷酸序列与GenBank数据库中A组鼻病毒(73血清型)同源性最高,为94.7%。结论该例重症肺炎患者为EB病毒和A组(73血清型)人鼻病毒混合感染,排除禽流感H5N1和SARS感染。     

含H5N1禽流感病毒核酸序列的病毒样颗粒的制备及应用

目的构建耐RNase酶的内含禽流感病毒H5N1亚型部分核酸序列的病毒样颗粒。方法构建中间载体pET32a-MS2,将禽流感病毒H5N1亚型的HA、NA和M基因片断连接到中间载体上,构建原核表达载体pET32a-MS2-HA,pET32a-MS2-NA和pET32a-MS2-M,分别转化宿主菌,诱导表达制备病毒样颗粒。结果表达载体经PCR、酶切鉴定和测序分析后证实构建成功。电镜观察到了病毒样颗粒,荧光定量RT-PCR试验表明颗粒稳定性良好。结论成功构建了含禽流感病毒H5N1部分核酸序列的病毒样颗粒且稳定性良好,可作为禽流感H5N1亚型RNA检测的标准品和质控品。     

SYBR Green I荧光定量PCR检测HIV-1前病毒方法的建立及应用

目的 建立SYBR Green I荧光定量PCR技术测定HIV-1前病毒载量方法,并以其观察相关病毒进入抑制剂对HIV-1前病毒的作用.方法 选择HIV-1的gag和env保守区域设计引物,建立SYBR Green I荧光定量PCR测定HIV-1前病毒载量的体系,并以其观察6 μg/ml～6 ng/ml的重组病毒巨噬细胞炎症蛋白（rvMIP）、逆转录酶抑制剂（AZT）、膜融合抑制剂（T-20）、rvMIP＋AZT、rvMIP＋T-20对HIV-1前病毒载量的影响.结果 所建立的荧光定量PCR体系的检测下限为101 Copies,其标准曲线的相关系数为0.998 9,斜率为-4.925,截距为35.70;除6 ng/ml的 rvMIP、AZT、T-20外,三种药物处理后HIV-1前病毒载量均显著减小（P〈0.05）,并呈质量浓度依赖性;联合用药者前病毒载量显著低于单用rvMIP者（P〈0.05）,HIV-1前病毒载量rvMIP＋T-20 〈rvMIP＋AZT〈rvMIP〈T-20〈AZT.结论 本研究建立的SYBR Green I荧光定量PCR检测方法具有灵敏、快速、准确及成本低廉等优点;与T-20或AZT联用可增强rvMIP对HIV-1前病毒的抑制作用.     

试验鸡感染H5N1亚型禽流感病毒后排毒规律的研究

目的　为揭示H5N1亚型禽流感病毒感染试验鸡后体内各组织的排毒规律。方法　将 180只 2 8d非免疫石岐杂鸡随机分为两组 ,每组 90只 ,一组为攻毒组 ,用含H5N1亚型AIV(A/Goose/GD/ 2 / 96 ,对 2 8d非免疫石岐杂鸡LD50 为 10 -5 4× 0 2ml)尿囊液 10 -4× 0 2ml/只经肌肉注射途径攻毒 ,一组接种 0 2ml/只灭菌生理盐水为对照。攻毒后前 4d ,每隔 6h观察试验组和对照组鸡的临床表现 ,每组随机抽取两只剖杀 ,收集脑、肝、脾、肾、胰腺、肌肉、喉头与泄殖腔拭子进行病毒分离 ;攻毒4d后改为每隔 12h取样 ,直至第 11d ,同时采集血清测定禽流感HI抗体效价。结果　试验鸡攻毒 18h后可从泄殖腔及喉头拭子中分离到病毒 ,持续到攻毒后第 7d左右 ;攻毒后 36h可以从脑、肝、脾、肾、胰腺的混合样品中分离到病毒 ,可持续到攻毒后第 10d左右 ;而肌肉组织中始终未分离到病毒     

福建省人感染HSN1禽流感病毒血凝素基因测定和分析

目的了解我省人感染高致病性禽流感H5N1亚型病毒血凝素基因的特点以及和其他毒株序列间的关系。方法从采集自两例福建省感染者的标本中扩增H5N1禽流感病毒血凝素基因并作序列测定，获得的序列同其他H5N1禽流感病毒的血凝素基因进行比较并作进化分析。结果获得福建省的两例感染者中的H5N1禽流感病毒血凝素基因，并推导出该基因的氨基酸序列。结果表明，两株病毒的血凝素基因在蛋白酶水解位点均含有多个连续的碱性氨基酸，符合高致病性禽流感病毒的特征。经Blast程序检索发现，我省两例感染者与国内其他省份的感染者携带的病毒血凝素基因具有很高的相似性，提示国内的H5N1禽流感病毒感染者的病毒来源可能有一定的关系。结论通过对两例福建省感染者标本中的H5N1禽流感病毒血凝素基因进行序列测定，了解病毒基因的部分特点。通过序列比较分析为分子流行病学调查提供线索，但还需对其他基因序列以及病毒的致病性等生物学特征作进一步分析。     

Clinical characteristics and distribution of genotypes of TT virus infection in a hepatitis C virus-hyperendemic township of a hepatitis B virus-endemic country (Taiwan)

BACKGROUND: The prevalence of TT virus (TTV) viremia, without definite clinical significance, has been reported to be higher among chronic hepatitis C patients. The status and clinical characteristics of TT virus (TTV) infection and distribution of TTV genotypes in a hepatitis C virus (HCV) hyperendemic township (Masago community) in a hepatitis B virus (HBV) endemic country (Taiwan) were investigated. METHODS: Sera from 100 Masago residents were tested for alanine aminotransferase (ALT) and markers of HBV, HCV and GB virus C/hepatitis G virus (GBV-C/HGV) and TTV-DNA. Sera of 250 blood donors as a control group were tested for TTV-DNA. Sera of Masago residents and blood donors with positive TTV-DNA were directly sequenced, and phylogenetic analyses were performed subsequently. RESULTS: The prevalences of TTV viremia in different age groups among individuals from Masago were significantly higher than that among blood donors. In regard to the subtypes of TTV, 23, seven, two, eight, one, six and one isolate were related to the genotypes 1a, 1b, 2a, 2b, 3, 4 and 5, respectively, from Masago and 21, 14, one, nine and three isolates were related to the genotypes 1a, 1b, 2a, 2b, and 4, respectively, from donors. No clinical or virological factor was associated with TTV viremia or TTV genotypes. CONCLUSIONS: TT Virus prevalence was higher among HCV hyperendemic township residents than blood donors with similar genotype distributions (genotype 1 was the most prevalent) in Taiwan. Neither TTV viremia nor a particular genotype was associated with HBV, HCV or GBV-C/HGV infection and abnormal ALT levels.     

临床肝病患者中庚型肝炎病毒(GBV-C/HGV)的感染

本文应用抗-HGV酶联免疫法(EIA)和逆转录套式聚合酶链反应法(RT-PCR)检测150份乙型、120份丙型、15份戊型和49份非甲-戊型肝炎患者血清。结果显示:乙肝、丙肝、戊肝和非甲-戊型肝炎患者中抗-HGV抗体的阳性率分别为22.0％(33/150)、25.0％(30/120)、33.3％(5/15)和40.1％(20/49)。其中乙型、丙型、戊型和非甲-戊型肝炎的抗-HGV抗体阳性者中,HGV RNA的阳性率分别为58.3％(7/12)、60.0％(6/10)、40.0％(2/5)和45.5％(9/12)。说明GBV-C/HGV可与HBV、HCV或HEV合并感染,该病毒可能引起临床型肝炎。     

{alpha}V{beta}3-integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2

HSVs enter cells in a receptor-dependent [nectin1 or herpesviruses entry mediator (HVEM)] fashion by fusion of the viral envelope with plasma membrane (neutral pH compartment), by endocytosis into neutral or acidic compartments, or by macropinocytosis/phagocytosis. The cellular determinants of the route of entry are unknown. Here, we asked what cellular factors determine the pathway of HSV entry. CHO cells lack β(3)-integrin and the respective α-subunits' heterodimers. We report that, in the absence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway independent of cholesterol-rich rafts and dynamin2. In the presence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway dependent on cholesterol-rich rafts and dynamin2. HSV enters J-nectin1 and 293T cells through a neutral compartment independent of cholesterol-rich rafts and dynamin2. α(V)β(3)-integrin overexpression in these cells modifies the route of entry to an acidic compartment dependent on cholesterol-rich rafts and dynamin2, hence similar to that in α(V)β(3)-integrin-positive CHO-nectin1 cells. In some cells, the diversion of entry from an integrin- and raft-independent pathway to an acidic compartment requiring cholesterol-rich lipids rafts and dynamin2 is irreversible. Indeed, HSV cannot infect CHO-nectin1-α(V)β(3) cells through any compartment when the αvβ3-integrin-dependent pathway is blocked by anti-integrin antibody, anti-dynamin2, or anti-acidification drugs. We conclude that the αvβ3-integrin is a determinant in the choice of HSV entry pathway into cells. Because the pathway dictated by αvβ3-integrin is through lipid rafts, the platforms for a number of Toll-like receptors, current findings raise the possibility that αvβ3-integrin acts as a sentinel of innate immunity.     

Selection of HSV capsids for envelopment involves interaction between capsid surface components pUL31, pUL17, and pUL25

During egress from the nucleus, HSV capsids that contain DNA (termed C capsids) are preferentially enveloped at the inner nuclear membrane over capsid types lacking DNA. Using coimmunoprecipitation and biochemical analyses of wild-type and mutant capsids, we identify an interaction between a complex of pU(L)17/pU(L)25, termed the C capsid-specific complex (CCSC), and pU(L)31, a component of the nuclear egress complex (NEC). We also show that the interactions between these components are dependent on expression of all three proteins but occur independently of the pU(L)31 interacting protein and NEC component pU(L)34, as well as a kinase encoded by U(S)3 that phosphorylates both pU(L)31 and pU(L)34. The interaction between the CCSC and pU(L)31 in the NEC suggests a mechanism to conserve viral resources by promoting assembly of only those viral particles with the potential to become infectious.     

2665例急性上呼吸道感染患儿的病原学及临床特征

目的 分析急性上呼吸道感染儿童患者的病原学及临床特征。方法 以南方医科大学珠江医院2009年11月至2015年9月收治的2 665例急性上呼吸道感染儿童为研究对象,采用qRT-PCR方法检测临床上常见的8种呼吸道病毒(流感病毒、呼吸道合胞病毒、副流感病毒、腺病毒、人类博卡病毒、人类冠状病毒、人类偏肺病毒、鼻病毒)。结果 共检测患儿标本2 665份,其中阳性标本1 566份,总阳性率为58.8%。四个季节中8种呼吸道病毒检出率存在明显差异,并以春季最高,夏冬季次之,秋季最低。儿童呼吸道病毒感染率随着年龄增加而逐渐降低,并以0~1岁婴幼儿病毒检出率最高64.5%。男童呼吸道病毒感染率高于女童,住院患儿呼吸道病毒检出率高于门诊患儿。混合感染标本260份,占阳性标本数的16.6%,主要集中于0~3岁儿童患者标本中,并因季节而异,秋冬季节较少,而春夏季节较为普遍。咳嗽为呼吸道病毒感染的主要临床症状,咳痰和流涕次之,临床症状在8种呼吸道病毒感染患儿中存在差异。结论 本调查分析了急性上呼吸道感染患儿中8种常见呼吸道病毒的病原学及临床特征,为指导临床治疗及防控提供相关数据。     

各型肝炎病毒单纯及重叠感染的研究

目的 探讨病毒性肝炎患者甲～戊，庚型肝炎病毒(HAV-HEV，HGV)单纯感染及重叠感染情况。方法 采用EIA法检测病毒性肝炎患者血清抗-HAV IgM，HBV标志物、抗-HCV IgM、抗-HDV IgM、抗-HEV IgM、抗-HGV IgM。结果 共检测210例病毒性肝炎患者HAV-HEV、HGV血清标志物，20例未检出(9．5％)，190例患者检出标志物阳性(90．5％)。HBV感染率89，5％(188／210，其中有34例为既往感染，占16．2％，现症感染154例，占73．3％)；HAV感染率29．0％(61／210)，HCV、HDV感染率均为8．1％(17／210)、HEV、HGV感染率依次为10．0％(21／210)、7．1％(15／210)。各临床类型中单纯感染占61．4％(129／210)，二重感染占32．4％(68／210)，以HAV HBV、HBV HDV、HBV HEV感染模式最常见，三重感染占6．2％(13／210)，以HAV HBV HDV感染模式最常见；临床上以肝炎肝硬化、重型肝炎重叠感染常见，急性肝炎最少见。结论 病毒性肝炎中HBV感染最常见，其次为HAV感染；单纯感染、二重感染多见，三重感染少见；重叠感染发生率随病情加重而增加。     

Interferon treatment of two patients with quadruple infection with hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis G virus (HGV), and TT virus (TTV)

Abstract: We administered interferon (IFN) to two patients who had quadruple infection with hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis G virus (HGV), and TT virus (TTV), a recently isolated novel DNA virus. Nine mega-units of natural alpha-IFN were administered daily during the first two weeks and thrice weekly during the following 22 weeks (total dose, 720 mega-units). In both cases, serum alanine aminotransferase (ALT) levels decreased during IFN administration but increased thereafter. The concentrations of HCV, HIV, HGV, and TTV declined with the administration of IFN. However, the concentrations of these 4 viruses increased after the cessation of IFN with the except of TTV in patient 2 which disappeared during treatment and did not subsequently reappear. IFN reduced the concentrations of 4 viruses, in an apparently independent manner.     

High Incidence of Zika or Chikungunya Infection among Pregnant Women Hospitalized Due to Obstetrical Complications in Northeastern Brazil—Implications for Laboratory Screening in Arbovirus Endemic Area

The diagnostic of arbovirus-related obstetric complications in high-risk pregnancy and childbirth care is challenging, especially in endemic areas. We conducted a prospective study to track active or recent Zika (ZIKV), dengue (DENV), or chikungunya (CHIKV) virus infection among hospitalized pregnant women (PW) with obstetric complications in a hospital at the epicenter of Zika outbreak and ZIKV-related microcephaly in Brazil. Clinical data and blood samples were collected at enrollment and 10 days after the admission of study participants, between October 2018 and May 2019. Further clinical data were extracted from medical records. Samples were screened by molecular and serological tests. Out of 780 participants, 93.1% (95% CI: 91.1–94.7%) presented previous DENV exposure (IgG). ZIKV, CHIKV, and/or DENV laboratory markers of recent or active infection were detected in 130 PW, yielding a prevalence of 16.6% (95% CI: 14.2–19.5%); 9.4% (95% CI: 7.4–11.7%), 7.4% (95% CI: 5.7–9.7%), and 0.38% (95% CI: 0.1–1.2%) of CHIKV, ZIKV, and DENV infections, respectively. Most ZIKV infections were detected by molecular assays (89.6%), while CHIKV infections were detected by serology (95.9%). Our findings highlight the need for arbovirus infections screening in PW with obstetrical complications, potentially associated to these infections in endemic areas regardless of the signs or symptoms suggestive of arboviral disease.