Effects of Naturally Occurring Six- and Twelve-Nucleotide Inserts on Newcastle Disease Virus Replication and Pathogenesis

Newcastle disease virus (NDV) isolates contain genomes of 15,186, 15,192 or 15,198 nucleotides (nt). The length differences reflect a 6-nt insert in the 5′ (downstream) non-translated region (NTR) of the N gene (15,192-nt genome) or a 12-nt insert in the ORF encoding the P and V proteins (causing a 4-amino acid insert; 15,198-nt genome). We evaluated the role of these inserts in the N and P genes on viral replication and pathogenicity by inserting them into genomes of two NDV strains that have natural genome lengths of 15,186 nt and represent two different pathotypes, namely the mesogenic strain Beaudette C (BC) and the velogenic strain GB Texas (GBT). Our results showed that the 6-nt and 12-nt inserts did not detectably affect N gene expression or P protein function. The inserts had no effect on the replication or virulence of the highly virulent GBT strain but showed modest degree of attenuation in mesogenic strain BC. We also deleted a naturally-occurring 6-nt insertion in the N gene from a highly virulent 15,192-nt genome-length virus, strain Banjarmasin. This resulted in reduced replication in vitro and reduced virulence in vivo. Thus, although these inserts had no evident effect on gene expression, protein function, or replication in vivo, they did affect virulence in two of the three tested strains.     

四株不同宿主来源的Class I基因3型新城疫病毒全基因组序列测定及比较分析

为了解析基因型相同但宿主来源不同的新城疫病毒的全基因组差异。本文采用RT-PCR方法分别获得4株（JS/3/09/Ch,ZJ/3/10/Ch,AH/2/10/Du,JS/9/08/Go）Class I 基因3型病毒的全基因组核苷酸序列,并与GenBank中已公布的Class I基因3型病毒全基因组序列进行比对分析。本实验4株病毒的基因组长度均为15198bp,在基因组1607～1608位有6碱基的缺失,在2381～2382位有12碱基的插入,裂解位点为112EQ/RQE/GRL117是标准弱毒特征。5株Class I基因3型病毒之间全基因组同源性超过93%;而与Class II弱毒株同源性最低只有72.2%;比较6个结构蛋白基因的同源性,NP基因的同源性最高(98.3%～96.4%),而P基因最低(96.1%～91.9%)。结果表明不同宿主来源的Class I基因3型新城疫病毒在遗传信息方面差异不大,但NP/F/L基因的变异幅度较P/M/HN基因明显。     

Complete Genome Sequence of Newcastle Disease Virus Mesogenic Vaccine Strain R2B from India

Mesogenic vaccine strains of Newcastle disease virus (NDV) are widely used in many countries of Asia and Africa to control the Newcastle disease of poultry. In India, the mesogenic strain R2B was introduced in 1945; it protects adult chickens that have been preimmunized with a lentogenic vaccine virus and provides long-lasting immunity. In this article, we report the complete genome sequence of the hitherto unsequenced Indian vaccine virus strain R2B. The viral genome is 15,186 nucleotides in length and contains the polybasic amino acid motif in the fusion protein cleavage site, indicating that this vaccine strain has evolved from a virulent virus. Phylogenetic analysis of this mesogenic vaccine virus classified it with the viruses belonging to genotype III of the class cluster II of NDV.     

Complete Genome Sequence of a Recombinant Marek's Disease Virus Field Strain with One Reticuloendotheliosis Virus Long Terminal Repeat Insert

Marek''s disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.     

Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.     

Complete genome sequencing and analysis of an anti-tumor Newcastle disease virus strain

HBNU/LSRC/F3, a Newcastle disease virus (NDV) strain stored in our lab, exhibited an anti-tumor ability in our previous studies. Nonetheless, very little is known about its genome sequence, which is vital for further study. Here, the complete HBNU/LSRC/F3 genome was fully sequenced and compared with other NDV sequences. Its genome contained 15,192 nucleotides (nt) consisting of two termini and six genes in the following order: 3′-Le-NP-P-M-F-HN-L-Tr-5′. Phylogenetic analysis indicated that this NDV strain belonged to the Class II genotype IX group. A multibasic amino acid (aa) sequence was found at the cleavage site (¹¹²RRQRR↓F¹¹⁷) within the fusion (F) protein, and a 6 nt insertion was present in the 5′ non-coding region of the NP gene. The whole genome sequence was highly similar to other genotype IX NDV genomes reported in China. Overall, this study provides insight into the sequence characteristics of genotype IX NDVs, which will be useful for subsequent investigations.     

Accumulation of a 3'-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells

Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome nearly 11 kb in length and is not formally thought to generate subgenomic RNA molecules during replication. Here, we report the abundant accumulation of a 3'-terminal 521- to 523-nucleotide (nt) genome fragment, representing a major portion of the 585-nt 3' untranslated region, in both mammalian (BHK-21) and mosquito (C6/36) cells infected with any of nine strains of JEV. In BHK-21 cells, the viral genome was detected as early as 24 h postinfection, the small RNA was detected as early as 28 h postinfection, and the small RNA was 0.25 to 1.5 times as abundant as the genome on a molar basis between 28 and 48 h postinfection. In C6/36 cells, the genome and small RNA were present 5 days postinfection and the small RNA was 1.25 to 5.14 times as abundant as the genome. The 3'-terminal 523-nt small RNA contains a 5'-proximal stable hairpin (nt 6 to 56) that may play a role in its formation and the conserved flavivirus 3'-cyclization motif (nt 413 to 420) and the 3'-terminal long stable hairpin structure (nt 440 to 523) that have postulated roles in genome replication. Abundant accumulation of the small RNA during viral replication in both mammalian and mosquito cells suggests that it may play a biological role, perhaps as a regulator of RNA synthesis.     

Generation by Reverse Genetics of an Effective,Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating,Highly Virulent Indonesian Strain

Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest that Ban/AF can provide better protection than commercial vaccines and is a promising vaccine candidate against NDV strains circulating in Indonesia.     

表达H5亚型AIV血凝素的重组NDV构建及免疫原性

利用反向遗传技术获得表达H5亚型禽流感病毒(AIV)血凝素(HA)的新城疫病毒(NDV)。克隆NDV clone 30的全长基因,通过在NDV的融合蛋白基因和血凝素-神经氨酸酶(HN)基因之间插入编码高致病性AIV分离株A/chicken/italy/8/98(H5N2)的血凝素基因开放阅读框从而获得两株重组新城疫病毒NDVH5和NDVH5m。NDVH5感染的细胞可以检测到两种HA转录产物。对于重组病毒NDVH5m,NDV位于HA ORF的转录终止信号序列被沉默突变消除,产生2.7个全长HA转录产物的折叠,从而使修饰过的HA得到稳定地高表达。1日龄小鸡的脑内接种证实了两种重组病毒均无致病性。鸡群在NDVH5m诱导产生的NDV和H5亚型AIV HA特异性抗体的免疫力下能够免于致死剂量的NDV与高致病性AIV的感染。血清学研究结果表明NDVH5m免疫鸡群产生的抗体可结合NP蛋白抗体的检测从而用于区分免疫和感染AIV的动物。因此,NDVH5m重组病毒可作为抗NDV和AIV的"二联疫苗",也可成为控制AJ的标记疫苗。     

The genome length of human parainfluenza virus type 2 follows the rule of six,and recombinant viruses recovered from non-polyhexameric-length antigenomic cDNAs contain a biased distribution of correcting mutations

Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the "rule of six." To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.     

FMDV OH99株基因组全序列的测定及其基因特征研究

采用RT-PCR方法对FMDV OH99株基因组全序列进行了分子克隆与测序。结果表明OH99株基因组全基因组序列长8040nt,其中5’NCR长1026nt,前导蛋白(L)编码区长603nt。该毒株结构蛋白与非结构蛋白编码区的核苷酸序列为6318nt,3’NCR长93nt,其后是poly(A)尾巴,测序结果表明该结构至少含有56个A。应用分子生物学软件,将OH99株与其它参考毒株进行了序列比较,并对其基因特征、推导的氨基酸序列进行了研究分析。结果显示,在分类地位上OH99株归属于O型FMDV,与OTY TW／97具有较高的同源性,而与其他参考毒株的差异性比较大,而且在基因组功能未知区域和3A编码区域具有两处明显的基因片段缺失现象,其中3A编码区缺失30nt,与OTY TW/97株相同,但功能未知区域的缺失状况与OTY TW／97稍有差异。根据VP1基因序列,对OH99株与参考毒株进行了系统发生树分析,分析结果表明OH99株与0TY TW／97株在同一基因型内,其遗传关系最近,而与其毒株遗传关系较远。     

Genotypic and pathotypic characterization of Newcastle disease viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have "died out" after the first panzootic (1926-1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India.     

Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.     

三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析。此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647~1648nt位。通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低。     

一株基因Ⅱ型新城疫强毒株的分子特性

从患病肉鸡群分离到一株新城疫病毒(NewcastleDiseasevirus,NDV)SQZ04。经蚀斑纯化后接种40日龄SPF鸡可诱发典型病变。经蚀斑纯化前和后的MDT为50·5h和51·2h,ICPI为2·0和1·92,IVPI为2·8和2·68,表明属强毒株。但F基因分型表明SQZ04属基因Ⅱ型,而且其与已知基因Ⅱ型的疫苗株LaSota、B1和Texas48的同源性分别为99·3%、98·7%和96·9%,显著高于与基因Ⅶ或Ⅸ型强毒株的同源性88·3%~88·6%或91·3%~92·1%。这是国内第一株属于基因Ⅱ型的NDV强毒株。SQZ04F多肽氨基酸裂解位点的序列为111GGRQGRL117,与弱毒株序列完全相同,这也是国内外首次报道具有这一氨基酸序列的强毒野毒株。然而,SQZ04株与其他已知强毒株的HN氨基酸同源性高达95·3%~97·3%,显著高于与弱毒株LaSota等的同源性87·8%~89·5%。     

Genetic Diversity of Newcastle Disease Virus in Wild Birds and Pigeons in West Africa

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d''Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d''Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.     

Genome comparison of a novel foot-and-mouth disease virus with other FMDV strains

The genome of a novel foot-and-mouth disease virus, HKN/2002, was 8104 nucleotides (nt) in length (excluding the poly(C) tract and poly(A) tail) and was composed of a 1042-nt 5'-untranslated region (UTR), a 6966-nt open reading frame, and a 93-nt 3'-UTR. Genome sequences of HKN/2002 and other known FMDV strains were compared. The VP1, VP2, and VP3-based neighbor-joining (NJ) trees were divided into distinct clusters according to different serotypes, while other region-based NJ trees exhibited some degree of intercross among serotypes. Mutations in HKN/2002 were revealed, including frequent deletions and insertions in the G-H loop of VP1, and deletion involving 10 amino acid residues in the 3A protein. An evolutionary relationship of HKN/2002 with an Asian FMDV lineage isolated from a Hong Kong swine host in 1970 was postulated. A 43-nt deletion identified in the 5'-UTR of HKN/2002 possibly contributed to the loss of one pseudo-knot domain.     

Molecular characterization of lapinized classical Swine Fever vaccine strain by full-length genome sequencing and analysis

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6-98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5'-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3'-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3'-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3'-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3'-UTR indicated that Indian CSFV strain requires further passage to obtain a 3'-UTR structure similar to most of the attenuated strains.