Molecular epidemiological investigation of foot-and-mouth disease virus in Korea in 2000

The genetic relatedness of 7 Korean type O field strains of foot-and-mouth disease virus (FMDV) in clinical specimens collected from 5 different geographic locations in 2000 was investigated. The sequence of 162 nucleotides (nt 478-639) at the 3' end of the 1D (VP1) genes was determined from amplified cDNA fragments, and subjected to the analysis for the sequence identity/divergence and phylogenetic relationship. The overall nucleotide sequence divergence among the 7 field strains was 0 to 3.8%, suggesting that they are closely related to each other. Phylogenetic analysis with the known Middle East-South Asia (ME-SA) topotype strains showed that the 7 Korean field strains formed two distinct clusters within the same lineage of the ME-SA topotype strains. Cluster 1 consisted of the strains of the primary foci of infection (Paju and Hongseong), and closely related to the strains prevailed in the Far East. Cluster 2 comprised those of subsequently affected regions (Boryeong, Yongin, and Chungju), and was further diverged from the Cluster 1. The result of phylogenetic analysis indicated that the Korean strains may have evolved from a common ancestor of the Pan Asia strains, and that at least 2 phylogenetically clustered variants within the same lineage were prevalent during the epidemic. The potential origin and sources of the virus introduction to Korea were discussed.     

Molecular epidemiology of serotype O foot-and-mouth disease virus isolated from cattle in Ethiopia between 1979-2001

Partial 1D gene characterization was used to study phylogenetic relationships between 17 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from Eritrea, Kenya, South and West Africa, the Middle East, Asia and South America. A homologous region of 495 bp corresponding to the C-terminus end of the 1D gene was used for phylogenetic analysis. This study described three lineages, viz. African/Middle East-Asia, Cathay and South American. Within lineage I, three topotypes were defined, viz. East and West Africa and the Middle East-Asia together with the South African isolate. The Ethiopian isolates clustered as part of topotype I, the East African topotype. Two clades (based on < 12 % nucleotide difference) A and B were identified within the East African isolates, with clade A being further classified into three significant branches, A1 (80% bootstrap support), A2 (89% bootstrap support) and A3 (94% bootstrap support). Clade B consisted of two Kenyan isolates. Within topotype I, the 17 Ethiopian isolates showed genetic heterogeneity between themselves with sequence differences ranging from 4.6-14 %. Lineage 2 and 3 could be equated to two significant topotypes, viz. Cathay and South America. Comparison of amino acid variability at the immunodominant sites between the vaccine strain (ETH/19/77) and other Ethiopian outbreak isolates revealed variations within these sites. These results encourage further work towards the reassessment of the type O vaccine strain currently being used in Ethiopia to provide protection against field variants of the virus.     

Antigenic variation among foot-and-mouth disease virus type A field isolates of 1997-1999 from Iran

The sequences of the antigenically relevant capsid proteins VP1-3 of 10 isolates obtained during an epizootic of serotype A foot-and-mouth disease virus in Iran, and collected within two and a half years, were found to be highly similar. However, each isolate differed by at least one amino acid from all others. This prompted us to analyze the immunological reactivity of the isolates. To this end, monoclonal antibodies (mAbs) against one isolate were generated and characterized with regard to neutralizing activity and reactivity with trypsinized virus. These mAbs as well as others raised against A22 virus were used for antigen profiling. This distinguished four antigenic conditions among the isolates and 16 reactivities among the mAbs. These findings, together with the observed sequence differences indicated the location of several epitopes. Many mAbs recognized the minor antigenic sites on VP2 and 3 and some the major site, the GH-loop of VP1. One epitope was composed of residues of the capsid proteins VP1 and 2.     

鸡新城疫病毒河北分离株F基因的分子特性分析

通过毒力测定、RT-PCR及F基因的序列测定与遗传进化分析,对2005-2008年从河北省部分地区的发病鸡群中分离到的10株新城疫病毒（NDV）进行了研究。各分离株经典毒力测定结果显示：MDT在37.6～54.4h之间,ICPI在1.71～2.0之间,IVPI值在2.16～2.8之间,均为新城疫病毒强毒株特征。F基因的序列测定表明,分离株之间的核苷酸序列具有77.4%～98.0%的同源性,与疫苗株Lasota的同源性为87.0%～98.9%,与国内标准强毒株F48E9同源性为89.7%～98.9%。推导其氨基酸序列分析表明,8个分离株的F蛋白的裂解位点氨基酸组成为112 R-R-Q-K-R-F117,具有NDV强毒株特征,与毒力测定结果相符,2个分离株的F蛋白的裂解位点氨基酸组成为112 G-R-Q-G-R-L117,与弱毒株特征相符。F基因分型和同源性比较显示：目前河北新城疫的流行以基因Ⅶ型为主（占70%）,同时兼有基因Ⅱ型（占20%）和基因Ⅸ型（占10%）。     

Molecular investigation of foot-and-mouth disease virus in domestic bovids from Gharbia,Egypt

An outbreak of foot-and-mouth disease (FMD) affecting cattle and water buffalo (Bubalus bubalis) occurred in Egypt during 2012/2013. The present study was undertaken to determine the current strains of the FMD virus (FMDV) and the prevalence of FMD among cattle and buffalo in Gharbia, Egypt. The diagnostic sensitivity of two RT-PCR assays for the detection of FMDV was evaluated. The results revealed that SAT2 was the causative agent. The percentage of infected of animals varied with the detection method, ranging from 62.5 % by the untranslated region (UTR) RT-PCR to 75.6 % by SAT2 RT-PCR. The overall prevalence and mortality rates were 100 and 21 %, respectively. The mortality was higher in buffalo (23.3 %) than it was in cattle (17 %). A partial sequence of SAT2 was identical (90–100 %) to Egyptian isolates and was close in similarity to sequences from Sudan and Libya. In conclusion, FMD in Egypt is caused by SAT2. No other serotypes were detected. The results of this study provided the valuable data regarding the epidemiology of SAT2 in cattle and water buffalo from Egypt, which strengthens the need to change the strategies of both control and prevention that help to prevent the spread of the disease.     

Molecular epidemiological studies on foot-and-mouth disease type O Taiwan viruses from the 1997 epidemic

Sequence diversity was assessed of the complete VP1 gene directly amplified from 49 clinical specimens during an explosive foot-and-mouth disease (FMD) outbreak in Taiwan. Type O Taiwan FMD viruses are genetically highly homogenous, as seen by the minute divergence of 0.2-0.9% revealed in 20 variants. The O/HCP-0314/TW/97 and O/TCP-022/TW/97 viral variants dominated FMD outbreaks and were prevalent in most affected pig-raising areas. Comparison of deduced amino acid sequences around the main neutralizable antigenic sites on the VP1 polypeptide showed no significant antigenic variation. However, the O/CHP-158/TW/97 variant had an alternative critical residue at position 43 in antigenic site 3, which may be due to selective pressure in the field. Two vaccine production strains (O1/Manisa/Turkey/69 and O1/Campos/Brazil/71) probably provide partial heterologous protection of swine against O Taiwan viruses. The type O Taiwan variants clustered in sublineage A1 of four main lineages in the phylogenetic tree. The O/Hong Kong/9/94 and O/1685/Moscow/Russia/95 viruses in sublineage A2 are closely related to the O Taiwan variants. The causative agent for the 1997 epidemic presumably originated from a single common source of type O FMD viruses prevalent in neighboring areas.     

Molecular epidemiology of foot-and-mouth disease virus type A in South America

A databank of 78 VP(1) complete sequences of type A foot-and-mouth disease virus (FMDV) from South American isolates was constructed. Forty-nine samples corresponded to FMDV that circulated between the years 1999-2008, mainly in Venezuela, where most type A outbreaks have occurred lately and twenty-nine to strains historically relevant for the continent. The phylogenetic analysis showed that all South American FMDV belonged to the Euro-SA topotype. Sixteen subgenotypes could be identified, based on a 15% nucleotide divergence cut-off criterion: eight are extinguished, three were active until the year 2002 and the remaining five circulated in Venezuela during the years 2001-2007, illustrating the potential for FMDV diversification under appropriate selective pressure. The last emergencies reported in already-free areas of Colombia in 2004 and 2008 were closely related to isolates acting in Venzuela. Evidence of positive selection over codon 170, within the immunogenic site 4 of VP1 protein, was recorded. A codon deletion in amino acid position 142, within the G-H loop, was found in some isolates within subgenotypes 14, 15 and 16. Conversely amino acid deletion 197 was restricted to all isolates within a particular genetic cluster. The present work is the first comprehensive phylogenetic analysis of FMDV type A in South America, filling a gap of knowledge with respect to both, historical and acting viruses. The results provided evidence that supports the ecosystem dynamics in the region, and also served as an input to establish genetic links of emergencies in already-declared free areas, highlighting the need for strengthening control activities.     

Molecular characterisation of Victorian Newcastle disease virus isolates from 1976 to 1999

OBJECTIVE: To characterise Newcastle disease virus isolates obtained in Victoria from 1976 to 1999 and identify the diversity of FO cleavage signal. DESIGN: RT-PCR using viral RNA extracted from positive NDV allantoic fluid was performed to amplify a segment of the NDV F and HN genes. Molecular characterisation of the nucleotide and amino acid sequences within the FO cleavage site was undertaken. RESULTS: All isolates contained 'avirulent FO cleavage signal sequence of varied amino acid composition. CONCLUSIONS: Molecular characterisation of past and present NDV FO cleavage signal sequences will provide valuable epidemiological information and assist in understanding the genetic origins and relationships of outbreaks.     

鸭肝炎病毒分子流行病学分析

为了解我国鸭病毒性肝炎(DH)的流行情况,本研究用标准DH病毒(DHV)Ⅰ型(DHV-1)抗血清和新型DHV(N-DHV)抗血清对国内分离的7株DHV进行了血清中和试验。结果表明,PLK株和YB3株为DHV-1;YB1、YB2、YB5、HY2和HY3株为N-DHV。同时,对国内分离的9株病毒的抗原相关VP1基因进行RT-PCR扩增和测序,对VP1基因的核苷酸与推导的氨基酸序列分析的结果表明,3株DHV-1的氨基酸序列同源性为95%左右,6株N-DHV的氨基酸同源性为98%左右;DHV-1分离株与N-DHV分离株间氨基酸同源性为72%～77%。参照同属微RNA病毒的人肠病毒的血清型分型标准,通过对DHV VP1基因序列同源性比较,判定9株病毒的血清型。依据VP1基因序列同源性进行的血清型分型结果与根据抗原性鉴定进行的血清型分型结果一致。同血清型病毒株间的VP1基因变异很小,表明DHV的抗原性稳定。同型不同毒株间VP1基因的差异可以导致病毒在致病力、细胞感染能力等方面的差异。研究结果表明,目前我国存在DHV-1和N-DHV两种独立的血清型,但尚未见血清亚型的流行。     

亚洲1型口蹄疫病毒L蛋白缺失株的构建及其生物学定性

为确定口蹄疫病毒(FMDV)L蛋白缺失对其生物学特性的影响,本研究在已建立的Asia1型FMDV反向遗传操作平台基础上,利用融合PCR方法缺失L蛋白基因编码区,构建了缺失L蛋白的重组Asia1型FMDV(rFMDV-ΔL)。拯救病毒的一步生长曲线结果表明,rFMDV-ΔL与亲本病毒相比在BHK-21细胞中的复制水平下降10倍,乳鼠毒力试验表明rFMDV-ΔL的毒力比亲本毒株下降6倍。另外,rFMDV-ΔL在BHK-21细胞上产生病变的速度明显变慢,形成蚀斑的时间延长,致乳鼠死亡所需时间也比亲本毒株明显延长。本研究是关于亚洲1型FMDVL蛋白结构与功能研究的首次报道,为亚洲1型FMDVL蛋白结构与功能的研究奠定了基础。     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Molecular characterization of avian paramyxovirus type 1 (Newcastle disease) viruses isolated from pigeons between 2000 and 2008 in Slovenia

Fourteen avian paramyxovirus type 1 (APMV-1; Newcastle disease) viruses isolated from dead free-living and domestic pigeons in Slovenia between 2000 and 2008 were analyzed by a molecular characterization of a part of the fusion protein gene, which included the region encoding the fusion protein cleavage site. Phylogenetic analysis indicated that the Slovene pigeon paramyxovirus type 1 (PPMV-1) viruses do not cluster together but instead are divided into two groups--4bi and 4bii--of sublineage 4b. Nine Slovenian strains were placed in group 4bii. Five other strains clustered together with PPMV-1 from group 4bi. The sequence of the fusion protein cleavage site of all Slovenian strains was typical for pathogenic APMV-1. The 112RRQKRF117 motif was present in the strains from group 4bii, whereas strains from group 4bi displayed the 112GRQKRF117 motif.     

An experimental infection in pigs using a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan

In this study, we carried out an experimental infection in pigs using a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan to analyze the clinical manifestation, antibody response and virus shedding patterns in pigs. We found that the virus was virulent in pigs, producing a synchronous disease in the inoculated pigs and efficient spread to direct contact pigs. These results are useful for epidemiologically investigating the 2010 epidemic in Japan and improving the measures for controlling possible future FMD outbreaks in Japan or elsewhere.     

鸵鸟新城疫病毒分子流行病学研究

应用RT-PCR、基因序列测定和分析以及鸡胚平均死亡时间(MDT)等方法,对1997～2000年间从中国北方分离的10株鸵鸟源新城疫病毒进行了F-糖蛋白基因序列测定、系统进化分析、基因分型和毒力测定。结果显示：10株鸵鸟源新城疫病毒分属Ⅱ、Ⅲ、Ⅵ、Ⅶ四个基因型。除1株为中发型毒株,其他均为速发型毒株；分析表明,引起鸵鸟发病的新城疫毒株有多种不同的来源。     

Immune responses to foot-and-mouth disease virus in pig farms after the 1997 outbreak in Taiwan

This paper reports on a retrospective study of the antibody responses to structural and non-structural proteins of FMD virus O Taiwan 97 in six pig herds in Taiwan in the year after the 1997 Taiwanese FMD outbreak. All herds were vaccinated against FMD after the outbreak as part of the countrywide control program. Three of the herds had confirmed FMD infections (herds N, O and P) and three herds remained non-infected (herds K, L and M). The serum neutralizing antibody titers and the non-structural protein ELISA (NSP) antibody responses in sows and 1-month-old pigs in the infected herds were higher than in the non-infected herds, but over time a number of positive NSP reactors were detected. From the serological studies and the herd monitoring and investigations it was considered that the FMD NSP positive reactors may not have constituted a true reservoir of FMD virus infection especially in herds where susceptible pigs were no longer present post-exposure or post-vaccination. Pigs vaccinated with an unpurified FMD type O vaccines being used at that time also showed false positive responses for NSP antibodies.