Genetic heterogeneity of SAT-1 type foot-and-mouth disease viruses in southern Africa

Summary.  Genetic relationships of 50 SAT-1 type foot-and-mouth disease viruses were determined by phylogenetic analysis of an homologous 417 nucleotide region encoding the C-terminal half of the VP1 gene and part of the 2A segment. Viruses obtained from persistently-infected African buffalo populations were selected in order to assess the regional genetic variation within the host species and compared with ten viruses recovered from recent and historical cases of clinical infection. Phylogenetic reconstructions identified three independently evolving buffalo virus lineages within southern Africa, that correspond with the following discrete geographic localities: (1) South Africa and southern Zimbabwe, (2) Namibia, Botswana and western Zimbabwe, and (3) Zambia, Malawi and northern Zimbabwe. This strict geographic grouping of viruses derived from buffalo was shown to be useful for determining the origin of recent SAT-1 epizootics in livestock. The percentage of conserved amino acid sites across the 50 SAT-1 viruses compared in this study was 50%. Most mutations were clustered within three discrete hypervariable regions, which coincide with the immunogenic G-H loop, H-I loop and C-terminus region of the protein. Despite the high levels of variation within the primary sequence, secondary structural features appear to be conserved. Received October 17, 2000/Accepted February 28, 2001     

Retrospective genetic analysis of SAT-1 type foot-and-mouth disease outbreaks in southern Africa

Summary. In areas where foot-and-mouth disease (FMD) is endemic in wildlife hosts, such as the Kruger National Park (KNP) in South Africa, control measures are in place that ensure that potentially infected antelope and buffalo do not come into close contact with domestic animals. In South Africa several SAT-1 outbreaks occurred nearly simultaneously in cattle and impala between 1971–1981. Phylogenetic analysis based on partial 1D gene nucleotide sequencing indicated that several of these outbreaks were linked and it is probable that disease spread from the intermediary impala antelope host to cattle in close proximity. Evidence was found for the involvement of viruses from a single KNP genotype in precipitating outbreaks in impala over a 10-year period. In addition, several unrelated outbreaks affecting cattle and impala occurred within a single year. Characterisation of outbreak strains from Botswana similarly revealed that a single genotype affected different species over a 10-year period and that transboundary spread of SAT-1 virus occurred on at least one occasion. This retrospective analysis of outbreak strains has clearly demonstrated that FMD control policies that address the role of antelope as intermediaries in disease transmission are crucial as these wildlife species play an important role in disease dissemination.     

Comparative genomics of serotype Asia 1 foot-and-mouth disease virus isolates from India sampled over the last two decades

This study deals with a comparative analysis of complete genome sequences of twenty-one serotype Asia 1 foot-and-mouth disease (FMD) field viruses isolated over a period of two decades from India, two vaccine strains and seven exotic sequences. The Indian viruses could be grouped in to three distinct lineages at the entire coding region, evolving independently probably under differential selection pressure as evident from the lineage-specific signatures identified. This comparison revealed 80% of amino acids at the polyprotein region to be invariant. Twenty-one residues in L, 3A and P1 region were identified to be under positive selection of which some are antigenically critical. Analysis at functionally crucial motifs, receptor contact residues, polyprotein cleavage sites and at putative T-cell epitopes expands the knowledge beyond other serotypes. Antigenic site II in betaB-betaC loop of VP2 was highly unstable suggesting its exposure to extreme immune pressure. A single cross-over at the L proteinase region in an isolate from buffalo, also featuring an extensive deletion at the 5' untranslated region (UTR), reflects the role of intraserotypic genetic recombination in natural evolution. The likely biological relevance of deletions/insertions observed at UTRs, VP1 and 3A could not be deduced. Altogether, a substantial amount of data raised on full length genomes of type Asia 1 virus adds value to the FMD virus genomics.     

Phylogenetic analysis of serotype A foot-and-mouth disease virus isolated in India between 1977 and 2000

Summary.  The genetic diversity among the Indian serotype A Foot-and-mouth disease virus (FMDV) isolates sampled over a period of 24 years (1977–2000) was studied by sequencing the VP1 gene. In the phylogenetic tree, constructed from 83 Indian and 37 other available sequences, the FMDV type A isolates were distributed into 10 major genotypes (designated as I–X). The Indian isolates were distributed in 4 genotypes (I, IV, VI and VII), and co-circulation of at least 2 genotypes (VI and VII) in different states of the country in recent years is evident from the result. The study also revealed differential geographic distribution of genotypes, for example, some (genotypes I and VII) were recovered from large geographical areas, some time even across the continents, suggesting the spread of the viruses beyond continental barriers. Localization of genotypes restricting to a particular country (genotypes III and X) was also observed in the study. The genetic diversity in the field isolates has been discussed in relation to the amino acid substitutions at the known antigenic sites. This work provides valuable insights into the epidemiological situation of type A FMDV in India and necessary information in the selection of suitable vaccine strain(s), if required, for the National FMD control program. Received August 28, 2001 Accepted November 19, 2001     

Partial sequence analysis of VP1 of Indian isolates of foot-and-mouth disease virus type Asia-1

Nucleotide sequence of 3' end of VP1 (1D region) was determined using RT-PCR amplified DNA of 31 foot and mouth disease virus (FMDV) type Asia-1 field isolates originating from 11 different geographically distinct states of India during the period 1987-2000. These field strains exhibited an average of 7.5% divergence among them and were found to be divergent from the Indian vaccine strains Asia-1 WBN 117/85, IND 8/79, and IND 63/72, by an average 5.9, 14.8, and 7.4% divergence, respectively. Phylogenetic analysis of these 31 field isolates including 3 of the vaccine strains of India and sequences of 22 Indian field isolates obtained from the GenBank revealed that all the Indian FMDV type Asia-1 isolates belonged to a single genotype comprising of two distinct lineages (Lineages A and B). All the field isolates under study belonged to the Lineage-B comprising 8 different clusters, which also includes the vaccine strains WBN-117/85 and IND 8/79. Surprisingly, another vaccine strain IND 63/72 formed Lineage-A. Phylogenetic analysis of sequences of another 23 exotic type Asia-1 isolates from 15 different countries obtained from the GenBank along with the 56 Indian isolates revealed the existence of three distinct genotypes. The prototype strain Asia-1 PAK 1/54 belongs to a separate genotype. Two strains from India along with one strain each from China and Russia belongs to another genotype. The third genotype is formed by the remaining isolates including all the 31 isolates from the present study and exotic viruses from 14 other different countries. Comparison of deduced amino acid (aa) sequence indicated that majority of the mutations were found within two distinct regions corresponding to amino acid positions 130-160 and 193-211. The motif at aa positions 138-141 in vaccine strains WBN 117/85, IND 8/79 and in all the field isolates was ETTS/P; however, the same motif in IND 63/72 was TQPT. The motif 153-156 in majority of Indian isolates including vaccine strains WBN 117/85 and IND 8/79 was LSGQ/R whereas the same motif seen in IND 63/72 was VSNR. The study revealed that the FMDV type Asia-1 isolates circulating in the country are not highly heterogeneous, but showed considerable genetic variations. Certain mutations were also observed in the residues, which have been proved to be contributing to the formation of neutralizing epitopes. In neutralization studies employing polyclonal antisera, type Asia-1 WBN 117/85 revealed broader serological spectrum than other vaccine strains of India used in this study.     

Virulence differences between monkeypox virus isolates from West Africa and the Congo basin

Studies indicate that West African and Congo basin isolates of monkeypox virus (MPXV) are genetically distinct. Here, we show Congo basin MPXV-ZAI-V79 is more virulent for cynomolgus monkeys as compared to presumed West African MPXV-COP-58. This finding may explain the lack of case-fatalities in the U.S. 2003 monkeypox outbreak, which was caused by a West African virus. Virulence differences between West African and Congo basin MPXV are further supported by epidemiological analyses that observed a similar prevalence of antibodies in non-vaccinated humans in both regions, while >90% of reported cases occurred in the Congo basin, and no fatal cases were observed outside of this region. To determine the basis for this difference in virulence, we sequenced the genomes of one human West African isolate, and two presumed West African isolates and compared the sequences to Congo basin MPXV-ZAI-96-I-16. The analysis identified D10L, D14L, B10R, B14R, and B19R as possible virulence genes, with D14L (ortholog of vaccinia complement protein) as a leading candidate.     

Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease

A panel of 12 monoclonal antibodies (MAbs) raised against foot-and-mouth disease virus (FMDV) of serotype C1 (FMDV C-S8c1) and 11 MAbs raised against other FMDVs have been used to evaluate the reactivity of 14 isolates of FMDV of serotype C1 (series FMDV C-S), 12 of them from one disease episode (Spain 1979-1982). The assays used were immunoelectrotransfer blot, immunodot and neutralization of infectivity. None of the isolates could be clearly distinguished by its reactivity with 6 non-neutralizing and 2 neutralizing MAbs raised against FMDV C-S8c1. In contrast, the isolates were distinguished in two groups by a 10(2)-fold difference in their reactivity with 6 neutralizing MAbs. The reactivity of MAbs with synthetic peptides indicated that conserved and non-conserved epitopes recognised respectively by neutralizing MAbs 4G3 and SD6 are localized in the immunogenic region (amino acids 138-156) of VP1. Thus, epidemiologically related FMDVs differ in at least one epitope critical for virus neutralization.     

Comparisons of the complete genomes of two Chinese isolates of a recent foot-and-mouth disease type Asia 1 virus

Summary China reported the first outbreak of foot-and-mouth disease (FMD) serotype Asia 1 in Chinese Hong Kong in March, 2005. Subsequently, this type of the virus was reported from mainland of China in April 2005. Up to September of 2006, it was detected in more than 15 areas of China. In this paper, the complete genomes of two Chinese isolates, Asia 1/HNK/CHA/05 and Asia 1/JS/CHA/05, of foot-and-mouth disease virus (FMDV) were sequenced and compared with some Chinese sequences and reference sequences from other countries. The identities between Asia 1/HNK/CHA/05 and Asia 1/JS/CHA/05 of 5′-UTR, L gene, P1 (VP1) gene, P2 gene, P3 gene, 3′-UTR are 84.8, 87.6, 86.4 (82.3%), 92.5, 92.8 and 95.3%, respectively. The data revealed that these two strains do not belong to the same genotype depending on the analysis of VP1 sequences, and neither of them have deleted bases in 5′UTR and 3A genes compared with the reference sequences. In addition, the secondary structures of their 5′UTR and 3′UTR are discussed.     

Genomic and antigenic characterization of viruses from the 1993 Italian foot-and-mouth disease outbreak

Summary. The origin and evolution of the type O foot-and-mouth disease viruses (FMDV) that caused the outbreak occurrence in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on the viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that these Italian viruses were grouped in the ME-SA topotype, closely related to viruses that circulated previously in the Middle East. The analysis of the nucleotide sequences in VP1, VP2 and VP3 showed a co-circulation during the epizootic of genetic variants, including viruses with amino acid replacements in VP3. For some of the isolates analyzed, values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.5 to 5.1 × 10⁻². The use of a panel of new monoclonal antibodies raised against an isolate from this outbreak, as well as monoclonal antibodies to FMDV O1-Switzerland 1965, showed differences in the reactivity pattern among some of the Italian isolates analyzed, which were consistent with the co-circulation of antigenic variants. These results support the potential for FMDV diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented.     

Interference between modified and virulent strains of foot-and-mouth disease virus

Summary Modified foot-and-mouth disease virus and interferon inhibited the multiplication and cytopathogenicity of virulent virus in bovine tissue cultures, but in cattle only modified virus inhibited the development of tongue lesions by virulent virus. Modified virus did not prevent generalisation of virulent virus after virus had been established suggesting that interference would only confer slight protection on cattle before development of immunity through antibody.     

Characterization of three infectious bursal disease virus isolates obtained from layer chickens in Iran

Three infectious bursal disease viruses (IBDVs) were isolated from field outbreaks in IBDV-vaccinated and non-vaccinated layer chicken flocks. Agar gel precipitation test (AGPT), immunoperoxidase staining, transmission electron microscopy (TEM), inoculation into embryonated eggs, and chicken embryo fibroblasts (CEFs) confirmed that the isolates were IBDVs. RT-PCR, restriction fragment length polymorphism (RFLP), and phylogenetic analysis demonstrated that the isolates were very virulent IBDV (vvIBDV) and showed a nucleotide sequence similarity of 96.3 to 99.8% in comparison with other vvIBDV strains. It was concluded that the Iranian isolates represented vvIBDV of serotype 1 originating from Europe, Japan, and China.     

Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African continent is lacking. Here, we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. However, similar analysis of the L region revealed random groupings of isolates from serotypes O and A. Comparisons between the phylogenetic trees derived from the structural coding regions and the L region pointed to a possibility of genetic recombination. The intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved, which likely reflects the selective pressures on these proteins. Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved. The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.     

Comparison of amino acid sequences at the amino acid 130-160 region of VP1 polypeptide of Indian field isolates of foot-and-mouth disease virus serotype Asia 1

The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.     

Phylogeny,genome evolution,and antigenic variability among endemic foot-and-mouth disease virus type A isolates from India

Summary. The capsid-coding (P1) and 3A regions of foot-and-mouth disease virus (FMDV) type A field isolates including two vaccine strains collected during 1977–2000 were analyzed. In the phylogenies, the isolates were distributed into two previously identified genotypes VI and VII, with multiple sub-genotypes that are temporally clustered. Comparison of the antigenic relationships of field isolates with the two vaccine strains (IND 17/77 and IND 490/97) and the reference strains of the genotypes VI (IND 233/99) and VII (IND 40/00) indicated two broad antigenic groups that correlate with the phylogenetic groupings (genotypes VI and VII), and are highly divergent from the vaccine strains. The maximum likelihood method of selection analysis identified a number of amino acid sites within the P1 region to be under weak positive selection. Some of the positively selected sites were mapped at/near the antigenically critical amino acid sites of the P1 region, indicating host immune pressure as one of the important driving force behind the observed genetic and antigenic diversity in FMDV. A small number of selected sites are located in the heparan sulphate-binding pocket of the virus, suggesting a fitness advantage for cell entry of the virus. No positive selection was detected in the 3A dataset.     

Genetic and antigenic variance of foot-and-mouth disease virus type Asia1

Summary.  The capsid protein encoding genes of five recent type Asia1 foot-and-mouth disease virus isolates, representative of three genotypes, were sequenced. The deduced amino acid sequences were aligned to each other and to two published sequences. The sequence differences suggested different antigenic properties of the isolates. One isolate was used to generate monoclonal antibodies (mAbs) which were analyzed for neutralizing activity and reactivity with trypsinized virus. Trypsin removes the major antigenic sites located at VP1. The five virus isolates formed three reaction patterns with the mAbs, irrespective of their genotype. Combination of all data allowed to suggest the location of the epitope of each antibody: the VP1 G-H and the VP2 B-C loop, the VP3 B-B knob, and the N-terminus of VP2, respectively, were involved. Received May 18, 1999/Accepted September 9, 1999     

Analysis of two VP60 capsid protein genes of rabbit hemorrhagic disease virus from viruses obtained from the same farm

Two distinct clones of the VP60 capsid protein gene of rabbit hemorrhagic disease virus were amplified from mixed liver tissue of rabbits collected from the same farm in the Xinjiang Uygur Autonomous Region of China in 2002. The results of DNA sequence analysis showed that the length of the VP60 gene in the first clone was 1,740 bp, similar to other VP60 genes. The length of the VP60 gene in the second clone was only 1,536 bp. The two clones were predicted to encode 579 and 511 amino acids, respectively.