甲型肝炎病毒细胞受体结构区氨基酸突变分析

利用ＰＣＲ引导的基因突变技术,对位于甲型肝炎病毒衣壳蛋白ＶＰ１上的细胞受体结合区进行氨基酸定点突变。结果发现当第１１４３,１１８７,１２０２和１２２５位氨基酸发生突变时,突变株病毒在细胞中的增殖动力学改变,病毒增殖量呈不同减少,提示这些位置上的氨基酸可能与细胞受体结合。     

脑心肌炎病毒VP1蛋白100位氨基酸的突变导致其对小鼠致病性的改变

作为脑心肌炎病毒(EMCV)的天然宿主,感染小鼠能引发多种疾病,其中包括脑炎、心肌炎及糖尿病.本研究利用之前构建完成的EMCV分离毒株BJC3的野生型感染性克隆,采用定点突变方法构建了4个VP1第100位氨基酸突变的突变株感染性克隆(VP1第100位氨基酸分别突变为丝氨酸、丙氨酸、异亮氨酸和脯氨酸)并获得了拯救病毒.虽然各突变病毒及野生型亲本拯救病毒在BHK-21细胞上形成的噬斑大小有所不同,但各病毒在BHK-21细胞上的复制水平未见差异.通过对突变病毒致病性进行系统分析,探究了VP1第100位氨基酸在病毒致病性及感染后疾病表型中所起的重要作用.结果表明,异亮氨酸和脯氨酸突变病毒对小鼠的致死率降低,脑中病毒载量减少且脑组织损伤轻微,而丝氨酸和丙氨酸突变病毒表现了与野生型亲本病毒相似的高致病性.结果证实,EMCVVP1第100位苏氨酸在病毒的体内复制中起重要作用,其突变会导致病毒对小鼠致病性的改变.     

脑心肌炎病毒VPl蛋白100位氨基酸的突变导致其对小鼠致病性的改变

作为脑心肌炎病毒（EMCV）的天然宿主,感染小鼠能引发多种疾病,其中包括脑炎、心肌炎及糖尿病．本研究利用之前构建完成的EMCV分离毒株BJC3的野生型感染性克隆,采用定点突变方法构建了4个VPl第100位氨基酸突变的突变株感染性克隆（VPt第100位氨基酸分别突变为丝氨酸、丙氨酸、异亮氨酸和脯氨酸）并获得了拯救病毒．虽然各突变病毒及野生型亲本拯救病毒在BHK-21细胞上形成的噬斑大小有所不同,但各病毒在BHK-21细胞上的复制水平未见差异．通过对突变病毒致病性进行系统分析,探究了VPl第100位氨基酸在病毒致病性及感染后疾病表型中所起的重要作用．结果表明,异亮氨酸和脯氨酸突变病毒对小鼠的致死率降低,脑中病毒载量减少且脑组织损伤轻微,而丝氨酸和丙氨酸突变病毒表现了与野生型亲本病毒相似的高致病性．结果证实,EMCVVPl第100位苏氨酸在病毒的体内复制中起重要作用,其突变会导致病毒对小鼠致病性的改变．     

PCV2 Rep基因启动子区vISRE突变株的生物学特性

摘要：【目的】为了初步揭示PCV2 Rep基因启动子区类干扰素刺激反应元件（vISRE）的生物学功能。【方法】应用感染性克隆技术构建了2株vISRE点突变的重组PCV2,对突变病毒在PK15细胞上的增殖特性、遗传稳定性及对干扰素刺激的反应特性进行了分析。【结果】Rep基因启动子区ISRE点突变后PCV2仍可在PK15细胞中正常复制,但病毒滴度比亲本毒株下降。PCV2 1740G-C在PK15细胞上3至10代之间遗传稳定,PCV2 1741A-T在PK细胞上第3代病毒保持突变基因的特征,但传至第7代时1743和1744位的AC突变为TT,并一直保持到第10代。100U/mL的PoIFN-α处理感染病毒的PK15细胞后,亲本毒株和2个突变毒株的阳性感染细胞数量均有增加,但亲本毒株病毒粒子数的增加显著高于2个突变毒株。【结论】Rep基因启动子区vISRE的突变影响PCV2在PK15上的增殖和对干扰素刺激的反应,推测其可能在干扰素促进病毒增殖中发挥调控作用。     

神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性的影响

近年来H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)茎部15~20个氨基酸的自发缺失时有报道,突变对于AIV生物学特性的影响还没有得到系统研究。应用反向遗传操作技术,拯救获得5株具有不同NA茎部长度的H5N1/PR8重组AIV。重组病毒的内部基因和血凝素(HA)基因来源相同,NA基因来源不同,并在NA茎部进行20个氨基酸的删除或添加突变。通过研究其生物学特性发现,5株重组病毒在SPF鸡胚中繁殖良好,其EID50、MDT和平均病毒滴度相似;NA茎部长短影响病毒的解凝能力,长茎病毒红细胞解脱能力比短茎病毒强;NA茎部15或20个氨基酸删除突变提高了重组病毒在MDCK细胞上的繁殖能力,短茎病毒释放出的病毒粒子数量是长茎病毒的10~100倍,释放时间提前6~10h,短茎病毒在MDCK细胞上形成的空斑也明显比长茎病毒的空斑大。实验结果揭示了AIV NA茎部氨基酸缺失突变的生物学意义,NA茎部15或20个氨基酸删除突变增强了AIV的细胞适应性,可能与现阶段H5N1亚型AIV宿主范围进一步扩大有关。利用反向遗传技术成功拯救了5株H5N1/PR8重组流感病毒,为流感病毒基因功能研究和重组疫苗研究建立了技术平台。通过对AIV NA茎部氨基酸的删除突变提高了病毒在MDCK细胞上的繁殖产量,为流感病毒细胞苗的生产提供了新的思路。     

江苏首例甲型H1N1(2009)流感病毒分离及其血凝素分子遗传特征分析

2009年6月12日,江苏确诊首例甲型H1N1(2009)病例。通过细胞和鸡胚分离系统,我们分离到一株具有较高血凝活性的病毒,命名为A/Jiangsu/1/2009。为了跟踪病毒的变异情况,我们开展了病毒的全基因组测序工作,在此基础上对其血凝素基因(Haemagglutinin,HA)的遗传特性进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Jiangsu/1/2009HA蛋白的有4个氨基酸发生了突变,但都不在已知的抗原位点上。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1的特点。与禽流感H1病毒相比,分离株HA蛋白受体结合位点上的E190D和G225D发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究首次对早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。     

动物细胞膜的分离纯化及其在病毒受体研究中的应用

细胞膜是动物细胞与胞外环境之间的屏障。病毒只有与细胞膜上的病毒受体特异性结合 ,才能进入细胞 ,进而启动其增殖周期。因此 ,病毒受体是病毒学研究的重要组成部分。分离纯化病毒受体所在的细胞膜作为病毒受体研究的实验材料 ,已经在许多病毒的研究中得到应用 ,并取得了很好的效果。现就动物细胞膜的分离纯化及其在病毒受体研究中的应用作一综述。     

细胞适应性流感病毒高产株的制备研究

与鸡胚培养制备的流感疫苗相比,细胞制备的疫苗具有免疫原性好、生产不受鸡胚限制等优点。但目前流感病毒株在细胞上产量较低,成为疫苗生产的主要限制因素。现就用于制备细胞适应性高产株主要的3种方法,即连续传代、随机突变构建病毒突变体和病毒重配的研究进展,以及突变位点对病毒增殖的影响作一概述。     

禽流感病毒H5N1血凝素基因适应性进化过程中的关键位点突变规律研究

目的:寻找导致禽流感病毒H5N1血凝素(HA)适应性进化的关键突变,建立氨基酸突变评价体系,对突变作用进行评估,印证它们与病毒适应性进化的关联性。方法:计算株频率和枝频率,寻找标记分枝,向根结点回溯寻找HA进化路径上的氨基酸突变。计算各突变位点氨基酸的频率变化、有效变换及高频次突变,基于以上几个因素建立突变评价体系。结果:建立了大规模自动化寻找突变的方法,计算得到HA进化过程中的氨基酸突变435个,通过氨基酸频率图表分析这些突变可以很好地反映病毒适应性进化过程,其中79个突变是有效变换,发生的位点为正选择位点,且多数位点落在HA抗原表位上;29个突变是高频次突变,其中多数也为有效变换,因而与病毒适应性进化密切相关。结论:大规模自动化寻找突变的方法可靠,建立的突变评价体系准确性高,找到的关键突变及位点对实验有很好的指导意义。     

人感染新型H10N3禽流感病毒分子溯源

【背景】1997年香港发生人感染禽流感事件以来,禽流感病毒成为持续威胁人类健康和公共卫生的重要病原体。【目的】对一例人感染新型H10N3禽流感病毒病例开展分子溯源研究。【方法】流感病毒分型检测采用RT-qPCR法,在下一代测序平台上完成病毒基因组测序,序列和系统进化分析采用BLAST和MEGA 6.1等生物信息学软件。【结果】2021年4月从严重呼吸道疾病患者体内分离到一株病毒,经核酸检测和序列分析,结果表明其为H10N3亚型禽流感病毒。从患者居所附近的农贸市场分离到一株基因高度同源的H10N3亚型禽流感病毒。分离株是一种新的基因重配H10N3禽流感病毒,其血凝素hemagglutinin(HA)和神经氨酸酶neuraminidase(NA)组合最早在2019年华东地区的家禽中检测到,6个内部基因来源于近年来中国南方家禽中流行的H9N2病毒。病毒的HA蛋白的裂解位点含有1个碱性氨基酸R,未插入多个碱性氨基酸,理论上不属于高致病性禽流感病毒。HA蛋白受体结合位点228位氨基酸残基由G突变为S,理论上增强了对人SAα2,6受体的亲和力。另外,未发现PB2蛋白E627K突变,但591位氨基酸...     

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.

Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.     

Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV.

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.     

The binding of hepatitis A virus to cell surfaces shows evidence of positive co-operativity

Abstract Radio-iodinated hepatitis A virus binds to cultured mammalian cells in a saturable manner, with about 1.4 × 10³ sites/cell and a S _0.5 of about 1.4 × 10⁻¹¹ M for FRhK-4 cells. This binding to FRhK-4 cells shows evidence of positive co-operativity, with a Hill coefficient of 2.1 (±0.1). This implies that the cellular receptor for the virus may have multiple binding sites and that the affinity of HAV for its receptor is increased if one of the binding sites is occupied by virus. Binding is completely blocked by two neutralising monoclonal antibodies, which also inhibit viral haemagglutination. A non-neutralising monoclonal antibody partially inhibits binding to FRhK-4 cells, but has no effect on haemagglutination.     

Amino acid substitutions in the V domain of nectin-1 (HveC) that impair entry activity for herpes simplex virus types 1 and 2 but not for Pseudorabies virus or bovine herpesvirus 1

The entry of herpes simplex virus (HSV) into cells requires the interaction of viral glycoprotein D (gD) with a cellular gD receptor to trigger the fusion of viral and cellular membranes. Nectin-1, a member of the immunoglobulin superfamily, can serve as a gD receptor for HSV types 1 and 2 (HSV-1 and HSV-2, respectively) as well as for the animal herpesviruses porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1). The HSV-1 gD binding domain of nectin-1 is hypothesized to overlap amino acids 64 to 104 of the N-terminal variable domain-like immunoglobulin domain. Moreover, the HSV-1 and PRV gDs compete for binding to nectin-1. Here we report that two amino acids within this region, at positions 77 and 85, are critical for HSV-1 and HSV-2 entry but not for the entry of PRV or BHV-1. Replacement of either amino acid 77 or amino acid 85 reduced HSV-1 and HSV-2 gD binding but had a lesser effect on HSV entry activity, suggesting that weak interactions between gD and nectin-1 are sufficient to trigger the mechanism of HSV entry. Substitution of both amino acid 77 and amino acid 85 in nectin-1 significantly impaired entry activity for HSV-1 and HSV-2 and eliminated binding to soluble forms of HSV-1 and HSV-2 gDs but did not impair the entry of PRV and BHV-1. Thus, amino acids 77 and 85 of nectin-1 form part of the interface with HSV gD or influence the conformation of that interface. Moreover, the binding sites for HSV and PRV or BHV-1 gDs on nectin-1 may overlap but are not identical.     

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.     

Neutralization of hepatitis A virus (HAV) by an immunoadhesin containing the cysteine-rich region of HAV cellular receptor-1

Hepatitis A virus (HAV) infects African green monkey kidney (AGMK) cells via the HAV cellular receptor-1 (havcr-1), a mucin-like type 1 integral-membrane glycoprotein of unknown natural function. The ectodomain of havcr-1 contains an N-terminal immunoglobulin-like cysteine-rich region (D1), which binds protective monoclonal antibody (MAb) 190/4, followed by an O-glycosylated mucin-like threonine-serine-proline-rich region that extends D1 well above the cell surface. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc portion of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVR-Fc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified through protein A-agarose columns. In a solid-phase assay, HAV bound to D1-Fc in a concentration-dependent manner whereas background levels of HAV bound to PVR-Fc. Binding of HAV to D1-Fc was blocked by treatment with MAb 190/4 but not with control MAb M2, which binds to a tag epitope introduced between the D1 and Fc portions of the immunoadhesin. D1-Fc neutralized approximately 1 log unit of the HAV infectivity in AGMK cells, whereas PVR-Fc had no effect in the HAV titers. A similarly poor reduction in HAV titers was observed after treating the same stock of HAV with murine neutralizing MAbs K2-4F2, K3-4C8, and VHA 813. Neutralization of poliovirus by PVR-Fc but not by D1-Fc indicated that the virus-receptor interactions were specific. These results show that D1 is sufficient for binding and neutralization of HAV and provide further evidence that havcr-1 is a functional cellular receptor for HAV.     

Hepatitis A virus inhibits cellular antiviral defense mechanisms induced by double-stranded RNA

The consequences of a hepatitis A virus (HAV) infection on cell-based antiviral responses and the interactions between virus and host cells resulting in persistent infections are poorly understood. In this report, we show that HAV does inhibit double-stranded (dsRNA)-induced beta interferon (IFN-beta) gene expression by influencing the IFN-beta enhanceosome, as well as dsRNA-induced apoptosis, which suggests that both effects may be connected by shared viral and/or cellular factors. This ability of HAV, which preserves the sites of virus production for a longer time, may allow the virus to establish an infection and may be the presupposition for setting up persistent infections. Our results suggest that the inhibitory effect of HAV on the cellular defense mechanisms might not be sufficient to completely prevent the antiviral reactions, which may be induced by accumulating viral dsRNA, at a later stage of infection. However, HAV seems to counteract this situation by downregulation of viral replication and in the following production of viral dsRNA. This ability of noncytopathogenic HAV acts dominantly on cytopathogenic HAV in trans. The downregulation might ensure the moderate replication which seems necessary for inhibition of the antiviral mechanisms by HAV and therefore for the persistent state of the HAV infection.