Effect of doubled dose administration of foot-and-mouth disease vaccine against heterologous virus infection in cattle.

Vaccination is a feasible approach for controlling foot-and-mouth disease (FMD). In FMD-free countries, vaccines are stored as a precautionary measure to control potential outbreaks. However, the challenge lies in pre-stocking optimal vaccines against the newly emerging strains. This study examined the potency of pre-stocked vaccines administered at elevated doses during emergencies. We vaccinated the cows with either a single or double trivalent vaccine dose containing two serotype O and one serotype A strains. Subsequently, vaccinated and unvaccinated cows were exposed to virulent strains of serotype O (O/JPN/2010; topotype Southeast Asia/Mya-98 lineage) or A (A/IRN/2016; topotype ASIA/G-VII lineage), which were genetically and antigenically distinct from the vaccine strains. Following challenge infections, all cows that received a single dose vaccination exhibited vesicular lesions with excreted viruses in the oral and nasal discharges. However, a substantial reduction was observed in the total clinical scores and virus titers in the sera and nasal discharges compared to those in the unvaccinated group. Cows receiving a doubled dose vaccination were completely protected from infection with O/JPN/2010 or demonstrated a significant decrease in viral shedding and clinical scores against A/IRN/2016. To note, vesicular lesions harbor significant amounts of viruses; thus, by mitigating their formation, viral transmission can be impeded, thereby slowing viral spread in the field. Furthermore, increasing the vaccine dose induced higher neutralizing antibody titers against heterologous strains. These findings suggest an alternative strategy for the effective management of future epidemics using pre-stocked vaccines.

Near-Complete Genome Sequences of Three Foot-and-Mouth Disease Virus O/ME-SA/Ind-2001e Isolates Obtained from Cattle and Pigs in Thailand in 2016.

Here, we report near-complete genome sequences of three foot-and-mouth disease viruses isolated in 2016 from bovine and porcine epithelial tissue samples collected in Nonthaburi, Songkhla, and Ratchaburi provinces, Thailand. These viruses were classified as serotype O, topotype ME-SA, and sublineage Ind-2001e.

Risk of transmission of foot-and-mouth disease by wild animals: infection dynamics in Japanese wild boar following direct inoculation or contact exposure.

Understanding of disease dynamics and viral shedding in wild boar and of the potential for disease spreading within wild boar and domestic pig populations is critical for developing effective control and eradication measures for foot-and-mouth disease (FMD). Accordingly, we infected experimentally wild boar and domestic pigs with FMD virus (FMDV) strains O/TAI/315/2016 and A/MOG/2013, and studied their susceptibility and viral transmissibility in both populations. Similar to FMDV-infected pigs, wild boar inoculated with both viruses exhibited vesicular lesions on their feet, snout, tongue and lip, although they did not show lameness. Further, inoculated wild boar were equally capable of transmitting the virus to all of their contact animals. While all contact pigs developed vesicular lesions after contact with inoculated animals, in contrast, no wild boar when exposed to the same infected animals showed obvious clinical signs. These results will be useful for further understanding of the critical roles in occurring and sustaining an FMD outbreak, and will be useful for establishing epidemiological surveillance programs and effective countermeasures for wild boar.

Administration of the antiviral agent T-1105 fully protects pigs from foot-and-mouth disease infection.

Foot-and-mouth disease (FMD) is a contagious disease affecting cloven-hoofed animals. Its transmissibility and antigenic variety make this disease difficult to control. Antiviral agents are expected to have an immediate effect that is independent of viral antigenicity; thus, they can serve as effective tools for inhibiting the spread of the causative agent, the FMD virus (FMDV), from infected animals. In this study, we investigated the antiviral activity of a pyrazinecarboxamide derivative, T-1105, against FMDV. Cytopathic effect inhibition assays revealed that T-1105 strongly inhibited the replication of 28 reference strains of all seven FMDV serotypes at non-cytotoxic concentrations. The antiviral effect of T-1105 against FMDV was also evaluated by experimental infection of domestic pigs. T-1105 was administered orally to pigs starting 1 h before or 6 h after the inoculation of a porcinophilic FMDV serotype O, topotype CATHAY. None of the pigs administered with T-1105 showed clinical signs of FMD. Moreover, no infectious FMDVs or FMDV-specific genes were detected in their sera, oral and nasal discharges, or tissues collected 48 h after virus inoculation. These findings strongly suggest that administration of T-1105 is effective in controlling the spread of FMDV in pigs.

Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan.

Foot and mouth disease (FMD) causes severe economic losses to the livestock industry of endemic countries, including Pakistan. Pakistan is part of the endemic pool 3 for foot and mouth disease viruses (FMDV), characterized by co-circulating O, A, and Asia 1 serotypes, as designated by the world reference laboratory for FMD (WRL-FMD). FMDV serotype A lineage ASIA/Iran-05 is widespread in buffalos and cattle populations and was first reported in Pakistan in 2006. This lineage has a high turnover, with as many as 10 sub-lineages reported from Pakistan over the years. In this study, we reconstructed the evolutionary, demographic, and spatial history of serotype A and one of its sub-lineages, A/ASIA/Iran-05/SIS-13, prevalent in Pakistan. We sequenced nearly complete genomes of three isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13. We estimated recombination patterns and natural selection acting on the serotype A genomes. Source and transmission routes in Pakistan were inferred, and the clustering pattern of isolates of the SIS-13 sub-lineage were mapped on a tree. We hereby report nearly complete genome sequences of isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13, along with purported recombinant genomes, and highlight that complete coding sequences can better elucidate the endemic history and evolutionary pressures acting on long-term co-circulating FMDV strains.

Quantitative analysis of infection dynamics of foot-and-mouth disease virus strain O/CATHAY in pigs and cattle.

Foot-and-mouth disease virus (FMDV) serotype O, topotype CATHAY is a known porcinophilic virus that has caused devastating damage to the pig industry. However, the minimum infectious dose via a natural infection route in pigs, the infection dynamics in cattle, and risk of viral transmission from infected cattle to pigs have not been quantitatively analyzed. The FMDV strain O/HKN/1/2015 was serially diluted and inoculated into pigs via an intraoral route to determine the infectious dose. We found that a 104.0 tissue culture infectious dose (TCID50) of the virus was insufficient, but 105.5 TCID50 was sufficient to infect pigs via the oral route. While cows inoculated with the strain showed increased temperature in their feet, typical clinical signs including vesicular development were not observed. The cows showed short-term and low levels of viremia and virus excretion only before the detection of virus neutralizing antibodies. FMDV genes were not detected in esophageal-pharyngeal fluid from cows after 14 days post inoculation. No genetic insertions that could be associated with host adaptation were observed in viruses isolated from infected cows. These findings indicate that cows infected with FMDV of O/CATHAY have a low risk of viral transmission or persistence. Information on the dynamics of virus infection is essential for ensuring the rapid and accurate diagnosis of this disease, and its surveillance.

Horizontal transmission of foot-and-mouth disease virus O/JPN/2010 among different animal species by direct contact.

Foot-and-mouth disease (FMD) is highly contagious and easily transmitted among species of cloven-hoofed animals. To investigate the transmission of FMD virus (FMDV) among different animal species, experimental infections using the O/JPN/2010 strain were performed in cows, goats and pigs. One cow or two goats/pigs were housed with a different species of inoculated animals, and clinical observations, virus shedding and antibody responses were analysed daily. Whilst all cows and goats were infected horizontally by contact with inoculated pigs, transmission from cows to goats/pigs and from goats to cows/pigs was not observed in all in-contact animals. In particular, no pigs were infected horizontally by contact with inoculated goats. Comparison with our previous study on experimental infections among animals of the same species indicates that horizontal transmission occurred more easily between animals of the same species than between those of the different species. These findings will be useful for establishing and performing species-specific countermeasures in farms and regions where multiple species of animals coexist in potential future outbreaks.

Development and evaluation of silver amplification immunochromatography kit for foot-and-mouth disease virus antigen detection.

A silver amplification immunochromatography (SAI) kit for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV)-FMDV-Ag SAI-was developed using the monoclonal antibody 1H5 recognizing the highly conserved N terminus region of VP2. The FMDV-Ag SAI can be used under conditions of high biosecurity containment as it does not require any apparatus. The FMDV-Ag SAI exhibited 10-100 times higher sensitivity against the five serotypes (O, A, Asia1, C, and SAT1) and similar sensitivity against SAT2 and SAT3, compared with the Svanodip® FMDV-Ag kit immunochromatography kit. The Svanodip kit showed inhibitory results with several saliva samples but not with the FMDV-Ag SAI kit. In a validation study using clinical samples (n = 132; vesicular epithelium = 92, vesicular lesion swabs = 20, saliva = 20) in Mongolia, the sensitivity of FMDV-Ag SAI in comparison with real-time reverse transcription-polymerase chain reaction revealed the following data: vesicular epithelium, 85.4% (76/89); vesicular lesion swab, 46.7% (7/17); and saliva, 36.8% (7/19). No cross-reactivity with the non-FMDV vesicular-forming viruses and taxonomically related viruses of the Picornaviridae family occurred. The FMDV-Ag SAI is a highly sensitive diagnostic tool that enables pen-side diagnosis without requiring the use of any equipment.

Genetic Determinants of Virulence between Two Foot-and-Mouth Disease Virus Isolates Which Caused Outbreaks of Differing Severity.

Individual foot-and-mouth disease virus (FMDV) strains reveal different degrees of infectivity and pathogenicity in host animals. The differences in severity among outbreaks might be ascribable to these differences in infectivity among FMDV strains. To investigate the molecular mechanisms underlying these differences, we estimated the infectivity of O/JPN/2000 and O/JPN/2010, which caused outbreaks of markedly different scales, in cell lines, Holstein cattle, and suckling mice. Viral growth of the two strains in cells was not remarkably different; however, O/JPN/2000 showed apparently low transmissibility in cattle. Mortality rates of suckling mice inoculated intraperitoneally with a 50% tissue culture infective dose (TCID50) of 10 for O/JPN/2000 and O/JPN/2010 also differed, at 0% and 100%, respectively. To identify genes responsible for this difference in infectivity, genetic regions of the full-length cDNA of O/JPN/2010 were replaced with corresponding fragments of O/JPN/2000. A total of eight recombinant viruses were successfully recovered, and suckling mice were intraperitoneally inoculated. Strikingly, recombinants having either VP1 or 3D derived from O/JPN/2000 showed 0% mortality in suckling mice, whereas other recombinants showed 100% mortality. This finding indicates that VP1, the outermost component of the virus particle, and 3D, an RNA-dependent RNA polymerase, are individually involved in the virulence of O/JPN/2010. Three-dimensional structural analysis of VP1 confirmed that amino acid differences between the two strains were located mainly at the domain interacting with the cellular receptor. On the other hand, measurement of their mutation frequencies demonstrated that O/JPN/2000 had higher replication fidelity than O/JPN/2010.IMPORTANCE Efforts to understand the universal mechanism of foot-and-mouth disease virus (FMDV) infection may be aided by knowledge of the molecular mechanisms which underlie differences in virulence beyond multiple topotypes and serotypes of FMDV. Here, we demonstrated independent genetic determinants of two FMDV isolates which have different transmissibility in cattle, namely, VP1 and 3D protein. Findings suggested that the selectivity of VP1 for host cell receptors and replication fidelity during replication were important individual factors in the induction of differences in virulence in the host as well as in the severity of outbreaks in the field. These findings will aid the development of safe live vaccines and antivirals which obstruct viral infection in natural hosts.

Experimental infection of pigs with a classical swine fever virus isolated in Japan for the first time in 26 years.

Following an outbreak of classical swine fever (CSF) in Japan, 2018, CSFV JPN/1/2018 was isolated from an infected pig sample. In this study, we carried out a comparative experimental infection in pigs using this strain and the highly virulent ALD strain and compared outcomes, including clinical manifestation, virus shedding patterns and antibody responses. Although pigs inoculated orally or intramuscularly with JPN/1/2018 developed hyperthermia and had decreased leucocyte numbers, they survived for the whole experimental period and showed less severe clinical signs than those infected with the ALD strain. We confirmed the presence of characteristic multifocal infarction of the margin of the spleen that arises following infection with JPN/1/2018, albeit that this finding was not observed in all infected pigs. Both viruses efficiently spread to contact pigs in a similar manner, suggesting in transmissibility between the two strains. Viral RNAs were detected in all clinical samples, especially whole blood samples, before the pigs developed hyperthermia until at least approximately 2 weeks after inoculation. Our findings will be valuable for the investigations into epidemic events occurring in Japan and for establishing diagnostic strategies and control measures against CSF.

Reverse transcription-PCR using a primer set targeting the 3D region detects foot-and-mouth disease virus with high sensitivity.

The potential of foot-and-mouth disease (FMD) to spread extensively means that rapid and accurate methods are needed for its diagnosis. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here, we designed the primer set FM8/9 to amplify 644 bases in the conserved 3D region of all seven serotypes of the FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with those using the primer set 1F/R, which targets the 5'-UTR, and real-time RT-PCR (rRT-PCR) assays described in the World Organization for Animal Health manual. Detection limits of the RT-PCR assays were determined for 24 strains, representing all serotypes. The sensitivities of RT-PCR assays using FM8/9 were 100.6 - to 103.8 -fold higher than those of 1F/R assays for 21 strains. To assess the validity of the methods for analysing clinical samples, sera and saliva samples collected daily from pigs and cows infected with FMDV were analysed using the four PCR assays. FM8/9 assays detected FMDV from all infected pigs and cows for longer periods than 1F/R assays, indicating that FM8/9 assays have higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

Pathogenesis of the attenuated foot-and-mouth disease virus O/JPN/2000 in experimentally infected pigs.

We examined the pathogenesis of the attenuated foot-and-mouth disease virus (FMDV) O/JPN/2000 in pigs. The virus used in this study was passaged three times in primary bovine kidney (BK) cells and once in baby hamster kidney-21 (BHK-21) cells after isolation. A plaque assay demonstrated that this virus exhibited the small plaque (SP) phenotype. There was no clinical or histological evidence of vesicular lesions in pigs intraorally inoculated with 106 50% tissue culture infectious dose (TCID50)/ml of the SP virus (SPV) of FMDV O/JPN/2000. Although fever was detected from 2 or 3 days post inoculation (dpi), there was no other prominent clinical sign up to 6 dpi. Virus shedding from saliva and nasal swab samples was not observed in any pigs inoculated with the SPV of FMDV O/JPN/2000. In the foot, mild lamellar degeneration of prickle cells in the upper layer of the stratum spinosum was histologically observed without development into vesicular or necrotic lesions. Immunohistochemical virus antigen- and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)-positive reactions observed in the foot at 1 dpi seemed to disappear after 3 and 6 dpi. Our findings suggest that the SPV of FMDV O/JPN/2000 had low pathogenicity against pigs by intraoral inoculation.

Comparative evaluation of two ELISA kits for detecting antibodies to a nonstructural protein of foot-and-mouth disease virus using serum samples collected from naturally and experimentally infected cows.

When foot-and-mouth disease (FMD) occurs and a "vaccination-to-live" policy is adopted in a country, the country must perform serological surveillance of a nonstructural protein (NSP) of FMD virus. The NCPanaftosa kit is the only kit for detecting antibodies to NSPs that is officially recognized as the reference regent by the World Organization for Animal Health; however, it is only used in South American countries. In this study, the specificity and sensitivity of the NCPanaftosa kit were compared with those of the PrioCHECK kit sold by an international company. Results in this study suggest that the PrioCHECK kit performs similarly to the NCPanaftosa kit in detecting antibodies to the NSP in the cattle population.

Early pathogenesis of the foot-and-mouth disease virus O/JPN/2010 in experimentally infected pigs.

We examined the histological distribution of the lesions and the viral antigen associated with the virus and virus RNA in multisystemic organs in the early stages of foot-and-mouth disease virus (FMDV) O/JPN/2010 infection in pigs. Characteristic lesions commonly observed in pigs with FMD arise following inoculation with 106 tissue culture infectious dose (TCID)50/ml of FMDV O/JPN/2010 in pigs at 3 days post inoculation (dpi) by a natural infectious route. However, none of the six pigs inoculated with 103 TCID50/ml of FMDV O/JPN/2010 showed any evidence of infection up to 6 dpi. Immunohistochemical detection for the FMDV antigen and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) showed that FMDV predominantly infected prickle cells in the stratum spinosum in the tongue, coronet and bulb of the heel, and caused these infected cells to undergo cell death by apoptosis. However, there was no evidence that FMDV O/JPN/2010 infected epithelial/epidermal basal cells in the basal layer. Epithelial lesions with viral antigen in the tongue were distributed in the dorsal surface but not in the papillae, corpus linguae or inferior surface of the tongue. Non-suppurative myocarditis and epithelial lesions in the esophagus with FMDV antigen were observed in all three pigs examined at 3 dpi.

Genome Sequence of Foot-and-Mouth Disease Virus of Serotype O Lineage Ind-2001d Isolated from Cattle in Mongolia in 2015.

We report the whole-genome sequence of the foot-and-mouth disease virus (FMDV) O/MOG/BU/2-7/2015 isolated in Mongolia in 2015. This virus is closely related to isolates identified in Southeast Asia in 2015 and is classified under the O/ME-SA/Ind-2001d lineage. This is the first detection of an FMDV of this lineage in Mongolia.

Genome variability of foot-and-mouth disease virus during the short period of the 2010 epidemic in Japan.

Foot-and-mouth disease virus (FMDV) is highly contagious and has a high mutation rate, leading to extensive genetic variation. To investigate how FMDV genetically evolves over a short period of an epidemic after initial introduction into an FMD-free area, whole L-fragment sequences of 104 FMDVs isolated from the 2010 epidemic in Japan, which continued for less than three months were determined and phylogenetically and comparatively analyzed. Phylogenetic analysis of whole L-fragment sequences showed that these isolates were classified into a single group, indicating that FMDV was introduced into Japan in the epidemic via a single introduction. Nucleotide sequences of 104 virus isolates showed more than 99.56% pairwise identity rates without any genetic deletion or insertion, although no sequences were completely identical with each other. These results indicate that genetic substitutions of FMDV occurred gradually and constantly during the epidemic and generation of an extensive mutant virus could have been prevented by rapid eradication strategy. From comparative analysis of variability of each FMDV protein coding region, VP4 and 2C regions showed the highest average identity rates and invariant rates, and were confirmed as highly conserved. In contrast, the protein coding regions VP2 and VP1 were confirmed to be highly variable regions with the lowest average identity rates and invariant rates, respectively. Our data demonstrate the importance of rapid eradication strategy in an FMD epidemic and provide valuable information on the genome variability of FMDV during the short period of an epidemic.

Experimental infections using the foot-and-mouth disease virus O/JPN/2010 in animals administered a vaccine preserved for emergency use in Japan.

The effectiveness of a vaccine preserved for emergency use in Japan was analyzed under experimental conditions using cows and pigs in order to retrospectively evaluate the effectiveness of the emergency vaccination performed in the 2010 epidemic in Japan. Cows and pigs were administered a vaccine preserved for emergency use in Japan at 3 or 30 days before virus infection (dbv) and were subsequently infected with the foot-and-mouth disease virus (FMDV) O/JPN/2010, which was isolated in the 2010 epidemic in Japan. All animals vaccinated at 30 dbv and one of three pigs vaccinated at 3 dbv showed no vesicular lesions during the experimental period. The virus titers and viral RNA loads obtained from clinical samples were lower in the vaccinated cows than in the non-vaccinated cows. The viral excretion periods were shorter in the vaccinated cows than in the non-vaccinated cows. In contrast, in the vaccinated pigs, the virus titers and viral RNA loads obtained from the samples, except for those obtained from sera, were not decreased significantly, and the viral excretion periods were not sufficiently shortened. These results suggest that the vaccine can protect against clinical signs of infection by the FMDV O/JPN/2010 in animals; however, it should be noted that in vaccinated and infected animals, especially pigs, clinical samples, such as saliva and nasal swabs, may contain excreted viruses, even if no clinical signs were exhibited.

Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus.

Further evaluation of an ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease virus.

An ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease (FMDV) was further evaluated using sequentially collected serum samples of experimentally infected animals, because the sensitivity of the kit used in a previous study was significantly low in field animals. The kit fully detected antibodies in infected animals without vaccination; however, the first detections of antibodies by the kit were later than those by the liquid-phase blocking ELISA that is used for serological surveillance in the aftermath of outbreaks in Japan, for detection of antibodies to structural proteins of FMDV. Additionally, although the kit effectively detected antibodies in infected cattle with vaccination, there were several infected pigs with vaccination for which the kit did not detect antibodies during the experimental period. Taken together, the kit may not be suitable for serological surveillance after an FMD outbreak either with or without emergency vaccination in FMD-free countries.