Cloning and Sequence Analysis of HN and F Protein Genes from a Strain of Goose Paramyxovirus

[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.

Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Cloning,Sequencing of F and HN Genes and Molecular Characteristics of Goose Paramyxovirus BZ02 Isolate

[ Objective] The aim of this study was to clone and sequence F and HN genes of the isolated goose paramyxovirus and thus further investigate on its pathogenesis and evolutionary origins. [ Method] According to the published F and HN gene sequences of Avian Paramyxovirus Typelat home and abroad, two pairs of primers （ FF, FR; HNF, HNR） were designed by DNAstar software, and Fand HNgenes were also amplified by PCR. The PCR products were recovered and ligated to T vector, while the positive clones were identified by ampicillin plate screening and PCR. After bacteria shaking, the plasmid was extracted and sent to Shanghai Sangon for sequencing. The sequences were compared with the sequences in GenBank, and the phylogenetic tree was drawn by DNAstar software. Meanwhile, the phylogenetic analysis of amino acid residues was also studied. [Result] The ORF of Fgene was 1 662 bp encoding 553 amino acids, and its cleavage site was 112R-R-Q-K-R-F117, which was consistent with the characteristics of virulent strains. 101-bit and 121-bit amino acid residues were K （Lys） and D （Asp）. The ORF of HN gene was 1 716 bp encoding 571 amino acids. The homology of HN sequence in 29 strains of goose paramyxovirus to YG97 was the highest, accounting for 99.6%. Analysis of glycosylation sites revealed that glycosylation site of BZ02 strain at 538 -540aa disappeared. The phylogenetic tree drawn by HN genes was highly consistent with that drawn by Fgenes. Compared with the homology of F and HN nucleotide sequence of the published typelgoose paramyxovirus in China, BZ02, JG97, HG97 and YG97 were divided into the same sub-group, and the nucleotide and amino acid sequences homologies were highest between BZ02 and YG97 strain. E Condusionl BZ02, JG97, HG97 and YG97 have the same evolutionary origin or all originate from YG97.

Discovery and characterization of novel paramyxoviruses from bat samples in China

Many paramyxoviruses are responsible for a variety of mild to severe human and animal diseases.Based on the novel discoveries over the past several decades,the family Paramyxoviridae infecting various hosts across the world includes 4 subfamilies,17 classified genera and 78 species now.However,no systematic surveys of bat paramyxoviruses are available from the Chinese mainland.In this study,13,064 samples from 54 bat species were collected and a comprehensive paramyxovirus survey was conducted.We obtained 94 new genome sequences distributed across paramyxoviruses from 22 bat species in seven provinces.Bayesian phylodynamic and phylogenetic analyses showed that there were four different lineages in the Jeilongvirus genus.Based on available data,results of host and region switches showed that the bat colony was partial to interior,whereas the rodent colony was exported,and the felines and hedgehogs were most likely the intermediate hosts from Scotophilus spp.rather than rodents.Based on the evolutionary trend,genus Jeilongvirus may have originated from Mus spp.in Australia,then transmitted to bats and rodents in Africa,Asia and Europe,and finally to bats and rodents in America.

野鸟源禽副黏病毒的流行病学调查及遗传进化分析

目的:调查我国野生鸟类中禽副黏病毒(Avian Paramyxoviruses,APMV)的流行和进化趋势。方法:通过RT-PCR、鸡胚分毒、血凝试验、遗传进化分析对野鸟源样品进行APMV流行病学调查和进化分析。结果:1219份野鸟样品中共检测出9份APMV阳性样品,APMV感染率为0.74%。其中APMV-1有8份,感染率为0.66%;APMV-4有1份,感染率为0.08%。鸡胚分毒成功分离出1株鸽源PPMV-1,遗传进化分析显示其属于基因VI.2.1.1.2.2型,与近年来国内流行的基因型一致。结论:野鸟在APMV的传播中具有重要作用,应加强对野鸟源PPMV-1的监测,研究结果对可能因跨种传播导致的公共卫生事件进行早期预警防控提供理论依据。

鹅副粘病毒不同分离株的血凝特性及LD_(50)比较

用分离鉴定的 11株鹅副粘病毒分别在 SPF鸡胚传代至 2～ 11代后，用鸡、鸭、鹅、鸽、鹌鹑五种禽类的红细胞作血凝试验，证明 11株病毒均不同程度地凝集五种禽类的红血球。同时， 10株病毒 LD50在 10－ 8～ 10－ 10之间。

三种检测禽副粘病毒2(型PMV-2)抗体方法的建立及其与HI方法的比较

建立了检测血清中禽副粘病毒2型(PMV-2)抗体的间接免疫荧光试验(IFA)、间接法酶联免疫吸附试验(ELISA)以及斑点酶联免疫吸附试验(Dot-ELISA)方法,并将三种方法同血凝抑制试验(HI)进行了比较。结果表明,建立的IFA、间接ELISA和Dot-ELISA等三种抗体检测方法其敏感性、特异性均很好,与HI方法相比,敏感性也大大增强。

普通棉耳狨猴副粘病毒自然感染的研究

对一个狨猴实验室中暴发急性呼吸道疾病进行了病因调查。通过动物剖检、病毒分离、电子显微镜检查和血凝及血凝抑制试验和动物感染实验 ,确定其病原是副流感病毒 型(仙台病毒 )。从而确定动物死亡原因是病毒引起肺炎感染造成。

The Mechanism of Henipavirus Fusion:Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses,represented by Nipah virus and Hendra virus,are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia,Southeast Asia,India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins;efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion(F) glycoprotein. The analysis of monoclonal antibody(mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein,which is a tetrameric oligomer,and not with soluble forms of G(sG) ,which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably,these mutants of G also appear to confer a post-receptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together,these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further,receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.

副粘病毒HN蛋白颈部与F相互作用区功能的研究

副粘病毒（Paramyxovirus）包膜上镶嵌着两种糖蛋白血凝素-神经氨酸酶（Hemagglutinin-neuraminidase,HN）和融合蛋白（Fusion protein,F）,两者的相互作用是决定病毒宿主范围、毒力和传播的关键。为探讨HN颈部与F相互作用区（Fusion interaction region,FIR）在膜融合机制中的作用,选取新城疫病毒（Newcastle disease virus,NDV）与人副流感病毒3型（human parainfluenza virus type 3,hPIV3）为研究对象,通过片段置换及同源重组技术构建嵌合体C1、C2,进一步将NDV及hPIV3HN的FIR内第51位丝氨酸（Serine,S）、第55位天冬氨酸（Aspartic acid,D）定点突变为丙氨酸（Alanine,A）,获得突变体NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A,对嵌合体及突变体蛋白的细胞表面表达效率、受体识别活性、神经氨酸酶活性、促细胞融合活性及半融合活性进行检测。结果：各嵌合体C1、C2及突变体NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A的细胞表达效率、神经氨酸酶活性（Neuraminidase,NA）与野生型相比差异不显著（P〉0.05）,但促细胞融合活性均有不同程度的降低（P〈0.05）,C1、C2、NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A分别为野生型的7%、9%、27%、19%、17%和21%;C1、C2、NDV S51A、NDV D55A、hPIV3S51A、hPIV3D55A的受体识别活性分别为14.7%、22.3%、35.5%、28.8%、33.9%和40.2%,与野生型相比差异显著（P〈0.05）。结果表明：副粘病毒HN蛋白颈部与F相互作用区的突变及置换使HN蛋白的促细胞融合活性、受体识别活性降低,其中第51位丝氨酸（S51）及第55位天冬氨酸（D55）发挥重要作用。

Detection of diverse viruses in alimentary specimens of bats in Macao

Bats carry a variety of viruses, and some of them cause public health problems. Macao, which is famous for its gambling industry, has a complex population structure. The globalization in such an international metropolis has enhanced the chance of disease transmission. Therefore, surveillance of zoonotic viruses is necessary for the early warning of potential emerging infectious diseases.Here, we report the first surveillance of bat viruses in Macao. In this study, we collected 1004 samples involving 10 bat species from 7 sites from April 2015 to May 2016, and examined the presence of viruses using nucleic acid-based methods. Coronaviruses, adenoviruses and paramyxoviruses were detected in these samples, with a high prevalence of coronaviruses. While,none was positive for hepatitis A virus, hepatitis E virus or hantavirus. Co-infections are not common in those bat species, but coronavirus HKU6 and adenovirus can be found commonly occurred in Myotis ricketti.

副粘病毒膜融合机制的研究进展

副粘病毒科的许多病毒可使人和动物致病。感染发生需要病毒包膜与宿主细胞膜的融合。一般来说,副粘病毒融合蛋白诱导的膜融合需要吸附蛋白的协同作用来实现。但是,吸附蛋白如何将受体信号传递给融合蛋白,并激活融合蛋白,引发细胞融合?其确切的机制尚不清楚,且不同的吸附蛋白激活融合蛋白的具体过程存在明显的差异性。本研究将对副粘病毒膜融合过程中的保守性与特异性进行综述,以期为研究该属病毒的膜融合机制研究提供线索。

Immune responses elicited by ssRNA(-) oncolytic viruses in the host and in the tumor microenvironment

Oncolytic viruses(OVs)are at the forefront of biologicals for cancer treatment.They represent a diverse landscape of naturally occurring viral strains and genetically modified viruses that,either as single agents or as part of combination therapies,are being evaluated in preclinical and clinical settings.As the field gains momentum,the research on OVs has been shifting efforts to expand our understanding of the complex interplay between the virus,the tumor and the immune system,with the aim of rationally designing more efficient therapeutic interventions.Nowadays,the potential of an OV platform is no longer defined exclusively by the targeted replication and cancer cell killing capacities of the virus,but by its contribution as an immunostimulator,triggering the transformation of the immunosuppressive tumor microenvironment(TME)into a place where innate and adaptive immunity players can efficiently engage and lead the development of tumor-specific long-term memory responses.Here we review the immune mechanisms and host responses induced by ssRNA(-)(negative-sense single-stranded RNA)viruses as OV platforms.We focus on two ssRNA(-)OV candidates:Newcastle disease virus(NDV),an avian paramyxovirus with one of the longest histories of utilization as an OV,and influenza A(IAV)virus,a well-characterized human pathogen with extraordinary immunostimulatory capacities that is steadily advancing as an OV candidate through the development of recombinant IAV attenuated platforms.