鸡传染性支气管炎病毒H株纤突蛋白基因的RT-PCR/RFLP分型

鸡传染性支气管炎病毒(IBV)属于冠状病毒科冠状病毒属,可引起鸡呼吸道、输卵管、肾脏、肠道及腺胃等多部位病变.近年来,由于新的IBV变异毒株不断出现,从而导致鸡传染性支气管炎病的不断爆发,造成严重的经济损失[1].IBV的基因组为单股RNA,约27.6kb,该基因主要编码3种主要结构蛋白:纤突蛋白(S)、膜蛋白(M)和核衣壳蛋白(N),其中S蛋白成熟裂解为S1和S2两个蛋白亚基.S1蛋白是IBV的主要免疫原基因,可刺激机体产生中和抗体,决定病毒的组织亲嗜性,在病毒血清学分类中起主要作用[1,2].H株是1993年在河南省分离的典型肾病变IBV毒株,我们对IBV的H株S1基因进行了RT-PCR及酶切分析,并将其PCR产物克隆入质粒载体,为进一步研究IBV的H株分子生物学特性和研制IBV基因工程疫苗打下基础.     

IBV广东分离株GD05 S1基因的克隆、鉴定及其表达

鸡传染性支气管炎病毒(Infectious Brochitis virus,IBV)属于冠状病毒科冠状 病毒属,可引起鸡呼吸道、输卵管、肾脏、肠道及腺胃等多部位病变.近年来,由于新的I BV变异毒株不断出现,从而导致鸡传染性支气管炎病的不断爆发,造成严重的经济损失 [1, 2]}.IBV的基因组为单股RNA,主要编码3种主要结构蛋白:纤突蛋白(S)、膜蛋白(M) 和 核衣壳蛋白(N),其中S蛋白成熟裂解为S1和S2两个蛋白亚基.S1蛋白是IBV的主要免疫原 基因,可刺激机体产生中和抗体,决定病毒的组织亲嗜性,在病毒血清学分类中起主要作用 [1,3].     

新城疫病毒F蛋白碱裂解位点修饰及外源基因的插入对 新城疫LaSota疫苗株致病力的影响

新城疫是危害养禽业发展的重要传染病.新城疫病毒(NDV)具有高度传染性和高致病性,融合蛋白(F)的F1/F2裂解位点存在多个碱性氨基酸并由此形成的泛组织嗜性一直以来被认为是NDV致病的主要决定因素.本研究利用已经构建NDV弱毒LaSota疫苗株反向遗传操作平台,将LaSota病毒F蛋白的碱裂解位点由GGRQGR↓L分别突变为GRRQRR↓F和GRRQRR↓L,在未加入TPCK胰酶的情况下分别成功拯救出突变修饰LaSota疫苗病毒株rL-FmF和rL-FmL,通过测定鸡胚平均致死时间(MDT)、脑内致病指数(ICPI)和静脉内致病指数(IVPI)等指标对其毒力进行评估,结果rL-FmF和rL-FmL,的ICPI值由LaSota的0.36分别上升为1.18和1.05,但.MDT均大于90小时,IVPI仍然均为0,表明碱裂解位点的突变可显著增强致病力.为了检测外源基因插入对病毒致病力的影响,进一步以rL-FmF为载体,分别构建并拯救出表达H5亚型禽流感病毒血凝素HA和增强绿色荧光蛋白EGFP基因的重组病毒rL-FmF-HA和rL-FmF-EGFP,经测定ICPI分别为0.67和1.10,但MDT均大于90小时,IVPI仍然均为0.结果表明,对rLaSota病毒F蛋白裂解位点2个非碱性氨基酸突变为碱性氨基酸,无论F2蛋白氨基端为F或L,均可显著增强其脑内接种致病力,接近中发型毒株标准,但对静脉内接种致病能力均无显著影响,而对鸡胚致死能力均保持rIaSota病毒缓发型特点(MDT≥90);外源基因的重组、表达可不同程度致弱病毒,其致弱程度与外源基因及其表达产物性质有关.结果提示,影响NDV致病力不仅仅局限于F蛋白裂解位点氨基酸序列;通过F裂解位点修饰及HA基因插入可以获得致病力较高但基本接近缓发型标准的重组病毒.     

英国用鸡痘苗弱毒载体制备抗鸡传染性支气管炎病毒基因工程疫苗获得成功

传染性支气管炎病毒(IBV)是鸡的重要病原体,属冠状病毒,具有大RNA基因组和囊膜。囊膜表面伸出的纤突蛋白质(Spike protein)(醣蛋白)能激发鸡产生保护性免疫力,病毒基因组中编码纤突基因的部位已弄清楚。     

禽Ⅰ型副粘病毒f基因克隆及序列分析

用RT-PCR一步法对云南省不同禽类(鸡、鸽子)3株禽I型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽I型副粘病毒各毒株同源性为88.1%～94.9%,与疫苗株LaSota和强毒株F48E9的同源性为85.6%.所分离两株新城疫病毒在F蛋白裂解位点区(112～117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株.鸽I型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMV ZQ98-1株在这一区域的序列完全相同,揭示为中强毒株.以1 662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒属于基因Ⅶ型,鸽I型副粘病毒属于基因Ⅵ型.     

禽I型副粘病毒f基因克隆及序列分析

用RT-PCR一步法对云南省不同禽类(鸡、鸽子)3株禽I型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽I型副粘病毒各毒株同源性为88.1%～94.9%,与疫苗株LaSota和强毒株F48E9的同源性为85.6%。所分离两株新城疫病毒在F蛋白裂解位点区(112～117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株。鸽I型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMV ZQ98-1株在这一区域的序列完全相同,揭示为中强毒株。以1 662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒属于基因Ⅶ型,鸽I型副粘病毒属于基因Ⅵ型。     

新城疫病毒ZJ1株F蛋白裂解位点突变对其融合活性的影响

新城疫病毒ZJ1毒株是近年来在我国水禽中流行并能引起水禽严重发病和死亡的强毒株,其F蛋白裂解位点有多个碱性氨基酸分布。将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸,构建了重组表达质粒pCI-FT。分别将突变前后的F蛋白与该毒株的HN蛋白在COS-1细胞共表达,表明突变前后的F蛋白均有融合活性;分别将突变前后的F蛋白与该毒株的HN蛋白在CEF细胞共表达,表明突变后F蛋白被裂解的活性大大降低。以上研究为下一步在全长cDNA克隆水平上对F蛋白裂解位点氨基酸序列进行相应突变,研究毒力相关因素以及构建毒力致弱疫苗株等奠定基础。     

禽Ⅰ型副粘病毒f基因克隆及序列分析

用RT—PCR一步法对云南省不同禽类(鸡、鸽子)3株禽Ⅰ型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽Ⅰ型副粘病毒各毒株同源性为88．1％--94．9％,与疫苗株LaSota和强毒株F48E9的同源性为85．6％。所分离两株新城疫病毒在F蛋白裂解位点区(112—117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株。鸽Ⅰ型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMVZQ98—1株在这一区域的序列完全相同,揭示为中强毒株。以1662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒届于基因Ⅶ型,鸽Ⅰ型副粘病毒届于基因Ⅵ型。     

鹅副粘病毒WF（01）G株F基因的测序与蛋白特性分析

用鹅副粘病毒WF01G分离株进行9-10日龄SPF鸡胚尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01G病毒的F基因,获得了1条长约1.7 kb的特异性条带。对PCR扩增产物测序。结果表明,扩增片段大小为1782 bp,含有1个1662 bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF01G株与其他7株鹅副粘病毒的同源性为84.8%-98.8%,与国内外其他NDV F基因的同源性为84.7%-93.8%,其中与国内标准强毒株F48E9的同源性为86.8%,说明WF01G与国内外的传统毒株有较大变异。与Tai-wan95株的同源性为93.8%,说明WF01G与Taiwan95株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-G ln-Lys-Arg-Phe117,表明为副粘病毒强毒株。蛋白疏水性和抗原性分析表明与标准强毒F48E9株相比没有太大的变异。     

新城疫病毒F和HN蛋白的氨基酸序列对其毒力的影响

新城疫病毒(Newcastle disease virus,NDV)属副粘病毒科副粘病毒属,为不分节的负极性单股RNA病毒,有囊膜.它虽只有一个血清型,但不同毒株的致病力差异较大,有强毒株、中毒株和弱毒株之分.业已证明,NDV不存在先天性控制病毒致病性基因,不同的毒株都含有相同的基因组和结构蛋白质组分.     

IBV青岛腺胃分离株（SD／97／02）S1蛋白基因的序列测定和分析

参考Genbank上发表的IBV S1纤突蛋白基因序列,设计了一对引物,对鸡传染性支气管炎病毒青岛腺胃分离株（SD／97／02）RNA进行RT－PCR扩增。将PCR产物克隆入pMD18-T载体中进行序列测定和分析。序列分析表明,该毒株的S1基因的G＋C％含量较少,为37.0%,存在HindⅢ,BamHⅠ,BglIⅠ,SacⅠ和SalⅠ位点,无EcoRⅠ位点,与其他毒株的同源性在87.02%－94.21%之间,在第154－429nt处为高度的变异区;将基因序列翻译成氨基酸后,假定的S1蛋白由540个氨基酸组成,等电点8.24,在蛋白质内部存在18个Cys,在S1与S2蛋白之间的剪切位点为HRRRR,这与大多数IBV毒株（RRF／SRR）不一样,有三个区域的氨基酸序列高度保守;169－181aa,230-250aa,485-506aa;与其他毒株进行抗原性比较后发现,在该毒株的320－326aa及390－401aa处的抗原表位消失,而在325－345aa、379－389aa处则出现了很强的抗原表位;第438－444aa处,其他IBV毒株（除ZJ971株外）原来存在的强抗原位点在本毒株中消失。在53－65位的氨基酸抗原性与其他毒株相比明显变弱。     

Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein.

Previous studies have identified two highly basic amphipathic helical regions in the human immunodeficiency virus type 1 transmembrane protein that, in vitro, display both cytolytic and calmodulin-binding and -inhibitory properties that could contribute to cellular dysfunctions and cytopathogenesis during a persistent viral infection. In the current study, the structural specificity of the cytolytic and calmodulin-binding activities of the human immunodeficiency virus type 1 lentivirus lytic peptide (LLP-1) are examined with synthetic peptide homologs and analogs. The results of these studies demonstrate that even minor changes in LLP-1 amino acid content can markedly affect these properties, suggesting that sequence variation in these highly conserved LLP sequences may correlate with alterations in viral cytopathic properties.     

鸡传染性支气管炎病毒纤突蛋白的目的基因点突变对其免疫原性的影响

反转录聚合酶链反应扩增鸡传染性支气管炎病毒中国流行株的主要免疫原纤突蛋白Ｓ１基因,将其插入载体ｐＵＣ１８的ＢａｍＨⅠ／ＨｉｎｄⅢ位点,在大肠杆菌中实现目的的基因的分子克隆。经克隆化Ｓ１基因的限制性酶切片多段多态性分析和Ｓｏｕｔｈｅｒｎ杂交之后,双脱氧链终止法测定其５‘端高变区核苷酸序列,并以此与ＧｅｎｅＢａｎｋ中的参考毒株Ｍａｓｓａｃｈｕｓｓｅｔｔｓ４１相应序列作比较,分析其同源性。     

Cooperative cleavage of the R peptide in the Env trimer of Moloney murine leukemia virus facilitates its maturation for fusion competence

The spike protein of murine leukemia virus, MLV, is made as a trimer of the Env precursor. This is primed for receptor-induced activation of its membrane fusion function first by cellular furin cleavage in the ectodomain and then by viral protease cleavage in the endodomain. The first cleavage separates the peripheral surface (SU) subunit from the transmembrane (TM) subunit, and the latter releases a 16-residue-long peptide (R) from the TM endodomain. Here, we have studied the distribution of R peptide cleavages in the spike TM subunits of Moloney MLV preparations with partially R-peptide-processed spikes. The spikes were solubilized as trimers and separated with an R peptide antibody. This showed that the spikes were either uncleaved or cleaved in all of its TM subunits. Further studies showed that R peptide cleavage-inhibited Env mutants, L(649)V and L(649)I, were rescued by wild-type (wt) Env in heterotrimeric spikes. These findings suggested that the R peptide cleavages in the spike are facilitated through positive allosteric cooperativity; i.e., the cleavage of the TM subunit in one Env promoted the cleavages of the TMs in the other Envs. The mechanism ensures that protease cleavage in newly released virus will generate R-peptide-cleaved homotrimers rather than heterotrimeric intermediates. However, using a cleavage site Env mutant, L(649)R, which was not rescued by wt Env, it was possible to produce virus with heterotrimers. These were shown to be less fusion active than the R-peptide-cleaved homotrimers. Therefore, the cooperative cleavage will speed up the maturation of released virus for fusion competence.     

Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin.

The ability of many viruses to replicate in host cells depends on cleavage of certain viral glycoproteins, including hemagglutinin (HA). By generating site-specific mutant HAs of two highly virulent influenza viruses, we established that the relationship between carbohydrate in the stalk and the length of the connecting peptide is a critical determinant of cleavability. HAs that lacked an oligosaccharide side chain in the stalk were cleaved regardless of the number of basic amino acids at the cleavage site, whereas those with the oligosaccharide side chain resisted cleavage unless additional basic amino acids were inserted. This finding suggests that the oligosaccharide side chain interferes with HA cleavage if the number of basic amino acids at the cleavage site is not adequate to nullify this effect. Similar interplay could influence cleavage of other viral glycoproteins, such as those of human and simian immunodeficiency viruses and paramyxoviruses.     

Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus

Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.     

中国1995～1999年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

应用RT-PCR方法扩增到了我国1995~1999年10株IBV现地分离株的核蛋白基因片段,并将其进行了克隆、序列测定及分析。结果发现,10株IBV分离株核蛋白基因均含有一个长1 230bp的ORF,编码由409个氨基酸残基组成的多肽,未发现碱基的插入和缺失。与GenBank中的20个IBV参考毒株核蛋白基因序列进行比较和分析,发现本研究分离的毒株主要分布于3个群中,该3群病毒主要包括我国IBV现地分离株。对n基因及其局部功能区序列比较发现,我国分离株与H120疫苗株N蛋白存在广泛的氨基酸变异。通过与s1基因系统发育进化树比较发现,我国IBV分离株存在基因重组现象。以上结果表明我国1995~1999年IBV毒株存在基因突变和基因重组现象。     

Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 4 Confer Increased Replication and Syncytium Formation In Vitro but Not Increased Replication and Pathogenicity in Chickens and Ducks

To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks.     

Making sense of spike D614G in SARS-CoV-2 transmission

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the etiologic agent of the current coronavirus disease 2019(COVID-19) pandemic, has evolved to adapt to human host and transmission over the past 12 months. One prominent adaptive mutation is the asparagine-to-glycine substitution at amino acid position 614 in the viral spike protein(D614G), which has become dominant in the currently circulating virus strains. Since spike protein determines host ranges, tissue tropism, and pathogenesis through binding to the cellular receptor of angiotensin converting enzyme 2(ACE2), the D614G mutation is hypothesized to enhance viral fitness in human host, leading to increased transmission during the global pandemic. Here we summarize the recent progress on the role of the D614G mutation in viral replication, pathogenesis, transmission, and vaccine and therapeutic antibody development. These findings underscore the importance in closely monitoring viral evolution and defining their functions to ensure countermeasure efficacy against newly emerging variants.     

Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease

In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.