A Spontaneously Occurring African Swine Fever Virus with 11 Gene Deletions Partially Protects Pigs Challenged with the Parental Strain.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 105 TCID50 of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact.

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD50)/ml, 101 HAD50/ml and 106 HAD50/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD50/ml and 101 HAD50/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD50/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 103 HAD50/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.

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.

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.

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.

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.

Development and Evaluation of a Rapid Antigen Detection and Serotyping Lateral Flow Antigen Detection System for Foot-and-Mouth Disease Virus.

We developed a lateral flow strip using monoclonal antibodies (MAbs) which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV). This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 10(3) to 10(4) of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden), which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs) showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvß6 for Foot-and-mouth disease virus isolation.

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)ß(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)ß(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)ß(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same.

Dose-dependent responses of pigs infected with foot-and-mouth disease virus O/JPN/2010 by the intranasal and intraoral routes.

Foot-and-mouth disease virus (FMDV) infection was successfully initiated in pigs by intraoral inoculation of both 10(6) and 10(3) TCID50 of FMDV O/JPN/2010 isolated from the 2010 epidemic in Japan. By intranasal inoculation, infection was established in pigs with 10(6) TCID50 of the isolate, but not with 10(3) TCID50 of the isolate. In the pigs inoculated with 10(6) TCID50 of the isolate, viruses and viral RNAs were obtained earlier from the pigs inoculated by the intraoral route than from the pigs inoculated by the intranasal route. These results support the theory that primary infection of a pig herd is more likely to occur by ingestion than by inhalation and that the oral cavity is likely to be a major entry route for FMDV in naturally exposed pigs.

Experimental infection of cattle and goats with a foot-and-mouth disease virus isolate from the 2010 epidemic in Japan.

In this study, we carried out experimental infections in cattle and goats using a foot-and-mouth disease virus (FMDV) isolate from the 2010 epidemic in Japan to analyze clinical manifestations, virus-shedding patterns and antibody responses in the animals. We found that the FMDV O/JPN/2010 isolate is virulent in cattle and goats, produces clinical signs, is spread efficiently by direct contact within the same species, and is persistently infectious in cattle. Quantitative analysis of levels of viral RNA in the tissues of cattle and goats infected with the isolate showed that the pharyngeal region is an important major target of the FMDV O/JPN/2010. Time course data of viral loads, excretion and transmission of the FMDV O/JPN/2010 in this study are key in providing quantitative data essential for epidemiological investigation and risk analysis in relation to disease controls.

Availability of a fetal goat tongue cell line ZZ-R 127 for isolation of Foot-and-mouth disease virus (FMDV) from clinical samples collected from animals experimentally infected with FMDV.

The availability of the fetal goat tongue cell line ZZ-R 127 for the isolation of Foot-and-mouth disease virus (FMDV) has not been evaluated using clinical samples other than epithelial suspensions. Therefore, in the current study, the availability of ZZ-R 127 cells for the isolation of FMDV was evaluated using clinical samples (e.g., sera, nasal swabs, saliva, feces, and oropharyngeal fluids) collected from animals experimentally infected with an FMDV isolate. Virus isolation rates for the ZZ-R 127 cells were statistically higher than those for the porcine kidney cell line (IB-RS-2) in experimental infections using cattle, goats, and pigs (P < 0.01). Virus titers in the ZZ-R 127 cells were also statistically higher than those in the IB-RS-2 cells. The availability of ZZ-R 127 cells for the isolation of FMDV not only from epithelial suspensions but also from other clinical samples was confirmed in the current study.

Foot-and-mouth disease virus antigen detection enzyme-linked immunosorbent assay using multiserotype-reactive monoclonal antibodies.

Monoclonal antibody (MAb)-based sandwich direct enzyme-linked immunosorbent assay (MSD-ELISA) methods that can detect foot-and-mouth disease virus (FMDV) antigens, both multiserotype (MSD-ELISA/MS) (for O, A, C, and Asia 1) and single-serotype (MSD-ELISA/SS) (for O, A, and Asia 1, specifically), were developed. MAb 1H5 was used as an antigen-trapping antibody that reacted with all seven serotypes of FMDV. The MAbs 71F2, 70C4, 16C6, and 7C2 were used as peroxidase-labeled detecting antibodies for multiple serotypes (O, A, C, and Asia 1), type O, type A, and type Asia 1, respectively, in both MSD-ELISA/MS and MSD-ELISA/SS. Our MSD-ELISAs showed high specificity. They produced a very low background of negative samples (buffer, plasma, and saliva) and were able to detect FMDV antigens from clinical samples (plasma and saliva), with results correlating with those of real-time reverse transcription-PCR. In terms of sensitivity, the MSD-ELISAs showed higher optical density values against each diluted serotype antigen than the indirect sandwich ELISA method, which is currently recommended in the manual of the World Organization for Animal Health. The sensitivity and specificity of the MSD-ELISAs seem to be sufficient for the antigenic diagnosis of FMDV.

Neutralizing monoclonal antibody sandwich liquid-phase blocking enzyme-linked immunosorbent assay for detection of Foot-and-mouth disease virus type O antibodies.

Liquid-phase blocking enzyme-linked immunosorbent assay (LPBE) using the neutralizing monoclonal antibody (mAb) sandwich method (M-LPBE) for detection of Foot-and-mouth disease virus (FMDV) type O antibodies was developed. Two neutralizing mAbs, 72C1 and 65H6, were raised against the FMDV O/JPN/2000 strain, and used as trapping and peroxidase-labeled detecting antibodies, respectively. Sera from animals experimentally infected with FMDV showed specific positive results by M-LPBE, which were correlated with the results of the virus neutralization test (VNT). When 303 negative bovine and 302 negative swine sera were tested, the specificity of M-LPBE was 100% and 99.7%, respectively. In addition, nine samples that had been collected in 2000 in Japan and regarded as evidently false positives by LPBE (supplied by the World Reference Laboratory for Foot-and-Mouth Disease) were uniformly negative by M-LPBE, just like VNT. Therefore, M-LPBE seems to have sufficient specificity for FMDV type O antibody screening and diagnosis.

Differentiation of foot-and-mouth disease virus-infected pigs from vaccinated pigs using a western blotting assay based on baculovirus-expressed nonstructural proteins 2C and 3D.

Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural proteins 2C and 3D were used as the antigens in a western blotting assay. This assay allowed for differentiation of FMDV-infected pigs from vaccinated pigs. Serial studies were performed using sera collected from experimentally infected and vaccinated pigs. Positive reactions were found in the sera derived from the experimentally infected pigs. There was, however, no positive reaction in the vaccinated pigs. These results suggest that this western blotting assay is a useful method for differentiation of infected pigs from vaccinated pigs.

Isolation of foot-and-mouth disease virus from Japanese black cattle in Miyazaki Prefecture, Japan, 2000.

Four outbreaks of foot-and-mouth disease (FMD) occurred from March to May 2000 in Miyazaki and Hokkaido Prefectures, Japan. FMD virus isolation was achieved by sampling probang materials from Japanese Black cattle in the third case found in Miyazaki Prefecture. The probang materials were inoculated to bovine kidney (BK) and bovine thyroid cell cultures. CPE was observed in the BK at two days post-inoculation. Specific amplified DNA segments for FMD virus (FMDV) were detected by reverse transcriptase polymerase chain reaction in the culture fluid. The FMDV was identified as type O by enzyme-linked immunosorbent assay (ELISA) for antigen detection and the nucleotide sequence encoding the VPI was determined. This FMDV is a strain that is widespread in Pan-Asia and was designated as O/JPN/2000 by the World Reference Laboratory of the Pirbright Institute, England. This report marks the first isolation of FMDV in Japan.