Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the "super" deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.     

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.     

以猪IgG重链恒定区为抗原载体的抗口蹄疫病毒DNA疫苗的研制

口蹄疫(Foot-and-Mouth Disease, FMD)是当今世界上最为严重的家畜传染病之一,主要危害猪、牛、羊等偶蹄动物.FMD的致病原为FMD病毒(FMDV),属小RNA病毒科口蹄疫病毒属,有A、O、C、SATⅠ、SATⅡ、SATⅢ及AsiaⅠ共7个血清型.FMDV结构较简单,完整的病毒颗粒由4种结构蛋白VP1、VP2、VP3及VP4各60个拷贝构成的衣壳包裹一条单股正链RNA组成,其中VP1是主要的抗原蛋白[1].     

VPg gene amplification correlates with infective particle formation in foot-and-mouth disease virus.

In order to analyze the function of VPg amplification in aphthoviruses, we have undertaken the first mutational analysis of the repetitive VPg-coding region using an improved foot-and-mouth disease virus (FMDV) cDNA clone from which infective viral RNA was synthesized. A set of VPg mutants was constructed by site-directed mutagenesis which includes different VPg deletion mutations, a VPg insertion mutation, and amino acid residue replacement mutations that interfere with binding of the VPg protein to the viral RNA and with its proteolytic processing. Our results revealed that an amazing flexibility in the number of VPgs is tolerated in FMDV. Optimal viability is given when three VPgs are encoded. Deletion as well as insertion of one VPg gene still resulted in infective particle production. Infective particle formation was observed as long as one VPg remained intact. No obvious differences in the individual VPg molecules with regard to their promoting viral RNA synthesis were observed, indicating that all three VPgs can act equally in FMDV replication. Mutant polyprotein processing was comparable to that of the wild-type virus. However, VPg mutants showed reduced viral RNA synthesis levels after infection. The levels of viral RNA synthesis and infective particle formation were found to correlate with the number of functional VPgs left in the mutant virus. These findings suggest a direct VPg gene dosage effect on viral RNA synthesis, with a secondary effect on infective particle formation.     

口蹄疫病毒三价复合多表位佐剂DNA疫苗构建及其免疫原性

以O型、A型口蹄疫病毒(FMDV)结构蛋白VP1全基因和AsiaI型FMDV两个基因拓扑型的结构蛋白VP1基因上的5个抗原表位基因作为主要免疫原基因,以来源于非结构蛋白3ABC和结构蛋白VP4上的3个Th2细胞表位基因作为辅助基因,构建了O型、A型和Asia1型FMDV复合多表位基因工程疫苗表达盒OAAT,在此基础上,以金黄色葡萄球菌肠毒素A(SEA)为基因佐剂,通过分子设计构建了SEA与OAAT融合表达基因。将构建好的表达盒OAAT与SEA融合表达基因克隆至真核表达载体PVAX1PCMV启动子下游,构建了口蹄疫三价基因佐剂DNA疫苗pEA。经Western blotting和IFA检测,目的蛋白在Hela细胞中获得正确表达。小鼠免疫实验表明,pA和pEA免疫组的血清抗体均能分别与O型、A型和AsiaI抗原反应,与对照组相比差异较显著,且pEA免疫组和灭活疫苗免疫组抗体水平均显著高于pA免疫组;同时pA和pEA免疫小鼠细胞因子IL-2、IFN-γ、IL-4和IL-10较对照组显著提高,且pEA免疫组的IL-2、IFN-γ和IL-4水平明显高于pA免疫组。用O/NY00和Asia1/YNBS/58株FMDV进行...     

Asia Ⅰ型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位.本研究设计和合成了由Asia Ⅰ型FMDV VP1蛋白136～160aa和198～211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因.利用BamH I、EcoR I和Xho I位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG.100μg FshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200 ID_(50)剂量FMDV攻击时得到了完全保护.由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防.     

Chimeric virus-like particles elicit protective immunity against serotype O foot-and-mouth disease virus in guinea pigs

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that can affect cloven-hoofed animal species, leading to severe economic losses worldwide. Therefore, the development of a safe and effective new vaccine to prevent and control FMD is both urgent and necessary. In this study, we developed a chimeric virus-like particle (VLP) vaccine candidate for serotype O FMDV and evaluated its protective immunity in guinea pigs. Chimeric VLPs were formed by the antigenic structural protein VP1 from serotype O and segments of the viral capsid proteins (VP2, VP3, and VP4) from serotype A. The chimeric VLPs elicited significant humoral and cellular immune responses with a higher level of anti-FMDV antibodies and cytokines than the control group. Furthermore, four of the five guinea pigs vaccinated with the chimeric VLPs were completely protected against challenge with 100 50% guinea pig infectious doses (GPID₅₀) of the virulent FMDV strain O/MAY98. These data suggest that chimeric VLPs are potential candidates for the development of new vaccines against FMDV.     

Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.     

Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong (2002)

The nucleotide sequence of the VP1 coding region of foot-and-mouth disease virus (FMDV) strain HKN/2002, isolated from a disease outbreak occurring in Hong Kong in February 2002, was determined and compared with the sequences of other FMDVs. The VP1 coding region was 639 nucleotides in length and encoded a protein of 213 amino acid residues. Comparison of the VP1 nucleotide sequence with those of other isolates indicated that HKN/2002 belonged to serotype O. A VP1-based sequence similarity tree of several South-east Asian FMDV-O isolates showed that HKN/2002 was most closely related to FMDV isolates found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Comparison of the amino acid sequence of the major immunogenic region of HKN/2002 with that of the serotype O vaccine strain, O1/Manisa/Turkey/69, reveals significant similarity, indicating that current serotype O vaccines may offer some degree of protection against HKN/2002.     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

口蹄疫病毒非结构蛋白P3区的基因序列及分析

以口蹄疫Akesu/ 5 8分离株的 5 3代牛舌皮病料为材料 ,采用RT PCR法 ,扩增和克隆了两个约 1.5kb的DNA片段。核酸序列测得结果对接后 ,涵盖了全部P3区的基因序列。口蹄疫Akesu/ 5 8分离株基因组P3区的核酸序列共计 2 ,72 4nt,包括一个终止密码子TAA ,共编码 90 7个氨基酸 ;其中非结构蛋白 3A的基因是 45 9nt,编码 15 3个氨基酸 ;3个 3B(VPg)基因分别是 6 9、72和 72nt,氨基酸分别为 2 3、2 4和 2 4;3C是 6 39nt,2 13个氨基酸 ;3D是1,413nt ,471个氨基酸。各蛋白间由Glu/Gly(Ser)连接。序列比较显示 :3A的C端易变 ,其它区的变易呈零星散在     

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.     

Recombinant Bivalent Vaccine against Foot-and-Mouth Disease VirusSerotype O／A Infection in Guinea Pig

Foot-and-mouth disease (FMD) is a highly contagiousdisease of cloven-hoofed animals such as cattle and pig.The disease causes explosive epidemics and heavyeconomic losses in the agriculture worldwide [1]. FMDvirus (FMDV) shows a high genetic and antigenicvariability, and has seven serotypes: O, A, C, AsiaI, SAT1,SAT2 and SAT3 [2]. The FMDV control is mainly imple-mented using chemically inactivated virus vaccines, whichmay contain residual living virus and pose a risk of virusreleas…     

The pvr1 locus in Capsicum encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg

Mutations in the eIF4E homolog encoded at the pvr1 locus in Capsicum result in broad-spectrum potyvirus resistance attributed to the pvr1 resistance allele, a gene widely deployed in agriculture for more than 50 years. We show that two other resistance genes, previously known to be eIF4E with narrower resistance spectra, pvr2(1) and pvr2(2), are alleles at the pvr1 locus. Based on these data and current nomenclature guidelines, we have re-designated these alleles, pvr1(1) and pvr1(2), respectively. Point mutations in pvr1, pvr1(1), and pvr1(2) grouped to similar regions of eIF4E and were predicted by protein homology models to cause conformational shifts in the encoded proteins. The avirulence determinant in this potyvirus system has previously been identified as VPg, therefore yeast two-hybrid and GST pull-down assays were carried out with proteins encoded by the pvr1 alleles and VPg from two different strains of Tobacco etch virus (TEV) that differentially infected Capsicum lines carrying these genes. While the protein encoded by the susceptible allele pvr1+ interacted strongly, proteins translated from all three resistance alleles (pvr1, pvr1(1), and pvr1(2)) failed to bind VPg from either strain of TEV. This failure to bind correlated with resistance or reduced susceptibility, suggesting that interruption of the interaction between VPg and this eIF4E paralog may be necessary, but is not sufficient for potyvirus resistance in vivo. Among the three resistance alleles, only the pvr1 gene product failed to bind m7-GTP cap-analog columns, suggesting that disrupted cap binding is not required for potyvirus resistance.     

猪口蹄疫病毒多抗原表位重组腺病毒的构建与鉴定

本研究设计构建了含有猪O型口蹄疫病毒VP1（21—60）-（141-160）-（200—213）位氨基酸的基因的重组腺病毒质粒pAd-VP,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化获得了重组腺病毒rAd—VP。该重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50为10^-10／mL。RT—PCR检测证明目的基因在mRNA水平上可有效表达;应用O型口蹄疫病毒标准阳性血清进行间接荧光抗体试验,在rAdVP感染的HEK-293A细胞的胞质可见清晰荧光。证明该重组腺病毒对VP1（21-60）-（141—160）-（200—213）位氨基酸的基因进行了成功的表达,从而为FMDV多抗原表位腺病毒活载体疫苗的研究奠定了基础。     

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

ABSTRACT: BACKGROUND: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. METHODS: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. RESULTS: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. CONCLUSIONS: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.     

Organization of 5S ribosomal RNA genes in tea (Camellia sinensis).

The 5S rRNA genes in the Camellia sinensis (L.) O. Kuntze (tea) genome are arranged as tandem repeat units of 300 and 325 bps. The 2 classes of tandem repeats were discovered by Southern hybridisation of tea genomic DNA with a 5S rRNA gene PCR product.     

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.     

An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle

To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.