Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Foot-and-mouth disease virus serotype SAT 3 in long-horned Ankole calf, Uganda.

After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest relatives isolated previously from buffalo in Uganda.

The N-terminal region (VP4) of the foot-and-mouth disease capsid precursor (P1-2A) is not required during its synthesis to allow subsequent processing by the 3C protease.

The capsid precursor (P1-2A) of foot-and-mouth disease virus is processed by the 3C protease (3Cpro) to VP0, VP3 and VP1 plus 2A. During capsid assembly, the VP0 is cleaved to VP4 plus VP2. Single amino acid changes in a conserved motif (YCPRP) near the C-terminus of VP1 can block processing of the capsid precursor by the 3Cpro, although the cleavage sites are located hundreds of amino acids distant from this motif, presumably due to misfolding. In contrast, we show here that the absence of the VP4 sequence during the synthesis of the capsid precursor does not affect its subsequent processing. Cleavage of this truncated precursor by 3Cpro at the VP3/VP1 and VP2/VP3 junctions occurred efficiently. Thus, in contrast to the presence of the YCPRP motif in VP1, there are no critical motifs near the N-terminus of the precursor, within VP4, required for correct cleavage by 3Cpro.

Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002-2009.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. Three different serotypes of the virus, namely O, A and Asia-1, are responsible for the outbreaks of this disease in these countries. In the present study, the nucleotide-coding sequences for the VP1 capsid protein (69 samples) or for all four capsid proteins (P1, seven representative samples) of the serotype A FMD viruses circulating in Pakistan and Afghanistan were determined. Phylogenetic analysis of the foot-and-mouth disease virus (FMDV) VP1-coding sequences from these countries collected between 2002 and 2009 revealed the presence of at least four lineages within two distinct genotypes, all belonging to the Asia topotype, within serotype A. The predominant lineage observed was A-Iran05 but three other lineages (a new one is named here A-Pak09) were also identified. The A-Iran05 lineage is still evolving as revealed by the presence of seven distinct variants, the dominant being the A-Iran05AFG-07 and A-Iran05BAR-08 sublineages. The rate of evolution of the A-Iran05 lineage was found to be about 1.2×10(-2) substitutions per nucleotide per year. This high rate of change is consistent with the rapid appearance of new variants of FMDV serotype A in the region. The A22/Iraq FMDV vaccine is antigenically distinct from the A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino acid substitutions, exposed on the virus surface, which may explain this antigenic difference.

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5' untranslated regions are efficiently translated in cells by a cap-dependent mechanism.

The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 5' untranslated region (5'UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.

Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination.

BACKGROUND: Foot-and-mouth disease (FMD) is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced. RESULTS: Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L), the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965) and serotype O viral isolate (K/117/1999) revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O. CONCLUSIONS: Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2), a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A and O has been obtained. In addition to characterization using the VP1 coding region, analyses involving the non-structural protein coding regions should be performed in order to identify evolutionary processes shaping FMD viral populations.

The role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda.

BACKGROUND: To study the role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda, serum samples were collected from 207 African buffalos, 21 impalas (Aepyceros melampus), 1 giraffe (Giraffa camelopardalis), 1 common eland (Taurotragus oryx), 7 hartebeests (Alcelaphus buselaphus) and 5 waterbucks (Kobus ellipsiprymnus) from four major National Parks in Uganda between 2005 and 2008. Serum samples were screened to detect antibodies against foot-and-mouth disease virus (FMDV) non-structural proteins (NSP) using the Ceditest® FMDV NS ELISA. Solid Phase Blocking ELISAs (SPBE) were used to determine the serotype-specificity of antibodies against the seven serotypes of FMDV among the positive samples. Virus isolation and sequencing were undertaken to identify circulating viruses and determine relatedness between them. RESULTS: Among the buffalo samples tested, 85% (95% CI = 80-90%) were positive for antibodies against FMDV non-structural proteins while one hartebeest sample out of seven (14.3%; 95% CI = -11.6-40.2%) was the only positive from 35 other wildlife samples from a variety of different species. In the buffalo, high serotype-specific antibody titres (≥ 80) were found against serotypes O (7/27 samples), SAT 1 (23/29 samples), SAT 2 (18/32 samples) and SAT 3 (16/30 samples). Among the samples titrated for antibodies against the four serotypes O, SAT 1, SAT 2 and SAT 3, 17/22 (77%; CI = 59.4-94.6%) had high titres against at least two serotypes.FMDV isolates of serotypes SAT 1 (1 sample) and SAT 2 (2 samples) were obtained from buffalo probang samples collected in Queen Elizabeth National Park (QENP) in 2007. Sequence analysis and comparison of VP1 coding sequences showed that the SAT 1 isolate belonged to topotype IV while the SAT 2 isolates belonged to different lineages within the East African topotype X. CONCLUSIONS: Consistent detection of high antibody titres in buffalos supports the view that African buffalos play an important role in the maintenance of FMDV infection within National Parks in Uganda. Both SAT 1 and SAT 2 viruses were isolated, and serological data indicate that it is also likely that FMDV serotypes O and SAT 3 may be present in the buffalo population. Detailed studies should be undertaken to define further the role of wildlife in the epidemiology of FMDV in East Africa.

Evidence for multiple recombination events within foot-and-mouth disease viruses circulating in West Eurasia.

Phylogenetic studies on foot-and-mouth disease viruses (FMDVs) circulating in the West Eurasian region have largely focused on the genomic sequences encoding the structural proteins that determine the serotype. The present study has compared near-complete genome sequences of FMDVs representative of the viruses that circulate in this region. The near-complete genome sequences (ca. 7,600 nt) were generated from multiple overlapping RT-PCR products. These amplicons were from FMDVs belonging to serotypes O, A and Asia-1, including members of the O-PanAsia-II and the A-Iran05 lineages, and of Group-II and Group-VII (Sindh-08) within serotype Asia-1, which are currently predominant and widespread in West Eurasia. These new sequences were analysed together with other sequences obtained from GenBank. Comparison of different regions of the FMDVs genomes revealed evidence for multiple, inter-serotypic, recombination events between FMDVs belonging to the serotypes O, A and Asia-1. It is concluded from the present study that dramatic changes in virus sequences can occur in the field through recombination between different FMDV genomes. These analyses provide information about the ancestry of the serotype O, A and Asia-1 FMDVs that are currently circulating within the West Eurasian region.

Dromedaries (Camelus dromedarius) are of low susceptibility to inoculation with foot-and-mouth disease virus serotype O.

Two sheep and five dromedaries were inoculated with a highdose of a cattle-passaged type O strain of foot-and-mouth disease virus (FMDV). The sheep developed typical FMD. The inoculated camels, which were placed in contact with five further dromedaries and four sheep, showed no visible signs of illness or vesicular lesions. However, one of them had a raised body temperature at 3 days post-inoculation (pi) and a viraemia from days 2 to 10; probang samples from this animal were negative for infectious virus, but a low level of FMDV RNA was detected in a sample taken on day 6 pi, five other samples taken from days 3 to 28 being negative. Examination of mouth swabs indicated a low level of FMDV RNA at days 1-5 pi in four of the five inoculated camels, but no infectious FMDV or FMDV RNA was detected in serum, probang or mouth swab samples from contact-exposed animals (camels and sheep). All the contact-exposed camels and sheep and two of the inoculated camels were serologically negative for FMD when tested up to day 28. In contrast, the camel with viraemia became serologically positive from day 14, and the other two inoculated camels (which had been exposed to FMDV in an earlier experiment) became serologically positive from day 10. The experiment suggested that dromedaries (1) are of low susceptibility to FMDV serotype O, (2) do not transmit infection, even by close contact, and (3) are unlikely to play a significant epidemiological role in FMD.

Genetic characterisation of the recent foot-and-mouth disease virus subtype A/IRN/2005.

BACKGROUND: According to the World Reference Laboratory for FMD, a new subtype of FMDV serotype A was detected in Iran in 2005. This subtype was designated A/IRN/2005, and rapidly spread throughout Iran and moved westwards into Saudi Arabia and Turkey where it was initially detected from August 2005 and subsequently caused major disease problems in the spring of 2006. The same subtype reached Jordan in 2007. As part of an ongoing project we have also detected this subtype in Pakistan with the first positive samples detected in April 2006. To characterise this subtype in detail, we have sequenced and analysed the complete coding sequence of three subtype A/IRN/2005 isolates collected in Pakistan in 2006, the complete coding sequence of one subtype A/IRN/2005 isolate collected during the first outbreak in Turkey in 2005 and, in addition, the partial 1D coding sequence derived from 4 epithelium samples and 34 swab-samples from Asian buffaloes or cattle subsequently found to be infected with the A/IRN/2005 subtype. RESULTS: The phylogenies of the genome regions encoding for the structural proteins, displayed, with the exception of 1A, distinct, serotype-specific clustering and an evolutionary relationship of the A/IRN/2005 sublineage with the A22 sublineage. Potential recombination events have been detected in parts of the genome region coding for the non-structural proteins of FMDV. In addition, amino acid substitutions have been detected in the deduced VP1 protein sequence, potentially related to clinical or subclinical outcome of FMD. Indications of differential susceptibility for developing a subclinical course of disease between Asian buffaloes and cattle have been detected.Furthermore, hitherto unknown insertions of 2 amino acids before the second start codon, as well as sublineage specific amino acids have been detected in the genome region encoding for the leader proteinase of A/IRN/2005 sublineage. CONCLUSION: Our findings indicate that the A/IRN/2005 sublineage has undergone two different paths of evolution for the structural and non-structural genome regions. The structural genome regions have had their evolutionary starting point in the A22 sublineage. It can be assumed that, due to the quasispecies structure of FMDV populations and the error-prone replication process, advantageous mutations in a changed environment have been fixed and lead to the occurrence of the new A/IRN/2005 sublineage. Together with this mechanism, recombination within the non-structural genome regions, potentially modifying the virulence of the virus, may be involved in the success of this new sublineage. The possible origin of this recombinant virus may be a co-infection with Asia1 and a serotype A precursor of the A/IRN/2005 sublineage potentially within Asian Buffaloes, as these appears to relatively easy become infected, but usually without developing clinical disease and consequently showing not a strong acute inflammatory immune response against a second FMDV infection.

Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus.

The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.

Reconstruction of the transmission history of RNA virus outbreaks using full genome sequences: foot-and-mouth disease virus in Bulgaria in 2011.

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.

Molecular characterization of serotype Asia-1 foot-and-mouth disease viruses in Pakistan and Afghanistan; emergence of a new genetic Group and evidence for a novel recombinant virus.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate appropriate vaccine selection and tracing of outbreaks. The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998-2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1 have circulated in Pakistan during this time. These are Group-II, -VI and, recently, a novel Group (designated here as Group-VII). This new Group has not been detected in neighbouring Afghanistan during the study period but viruses from Groups I and -II are in circulation there. Using near complete genome sequences, from FMD viruses of serotypes Asia-1 and A that are currently circulating in Pakistan, we have identified an interserotypic recombinant virus, which has the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05(AFG-07) sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses.

Genetic diversity of foot-and-mouth disease virus serotype O in Pakistan and Afghanistan, 1997-2009.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan; serotypes O, A and Asia-1 of the virus are responsible for the outbreaks in these countries with FMDV type O usually being the most common. In the present study, the nucleotide sequences encoding the FMDV capsid protein VP1 from virus samples were determined. Phylogenetic analysis of the serotype O FMD viruses circulating in Pakistan and Afghanistan between 1997 and 2009 revealed the presence of at least three different lineages within the ME-SA (Middle East South Asia) topotype. The three lineages detected in this study are Pak98, Iran2001 and PanAsia. The PanAsia lineage is currently dominant in the area and is evolving with time as revealed by the appearance of distinct variants e.g. PanAsia-II and a new variant designated here as PanAsia-III. The rates of evolution of the O-PanAsia-II and III sublineages prevalent in the region were found to be 6.65 × 10(-3) (95% CI=5.49-7.80 × 10(-3)) and 7.80 × 10(-3) (95% CI=6.72-8.89 × 10(-3)) substitutions per nucleotide per year, respectively. The present study reveals the presence of multiple (sub-)lineages of FMDV serotype O co-circulating in the region and that significant new variants are frequently emerging.

Dynamics of picornavirus RNA replication within infected cells.

Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks the initiation of negative-strand synthesis. We have now examined the dynamics of RNA replication, measured by quantitative RT-PCR, within cells infected with either swine vesicular disease virus (an enterovirus) or foot-and-mouth disease virus as regulated by the presence or absence of guanidine. Following the removal of guanidine from the infected cells, RNA replication occurs after a significant lag phase. This restoration of RNA synthesis requires de novo protein synthesis. Viral RNA can be maintained for at least 72 h within cells in the absence of apparent replication but guanidine-resistant virus can become predominant. Amino acid substitutions within the 2C protein that confer guanidine resistance to swine vesicular disease virus and foot-and-mouth disease virus have been identified. Even when RNA synthesis is well established, the addition of guanidine has a major impact on the level of RNA replication. Thus, the guanidine-sensitive step in RNA synthesis is important throughout the virus life cycle in cells.

Analysis of the epidemiological dynamics during the 1982-1983 epidemic of foot-and-mouth disease in Denmark based on molecular high-resolution strain identification.

An epidemic of foot-and-mouth disease (FMD) causing a total of 23 cases in 1982-1983, primarily on the island of Funen, Denmark, was subjected to molecular epidemiological investigations. In an attempt to exploit the quasi-species nature of foot-and-mouth disease virus strains for molecular high-resolution strain identification in order to analyse the dynamics of this epidemic, full-length VP1 coding regions were sequenced for 17 isolates collected at different farms during the epidemic. The sequence information together with epidemiological information gathered during the epidemic suggests that the epidemic was caused by at least three introductions across Danish borders and one case of airborne transmission between two islands in Denmark over a distance of 70 km. The assortment of nucleotide markers among the three strains is indicative of common recombination events in their evolutionary history, and the prerequisite of co- or superinfection of animals with variant strains in turn implies that they have a common source or epidemiologically related sources originating from an area with endemic FMD.