African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping.

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.

A previous study proved that vGPE⁻ mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE⁻ vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE⁻ vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein E^rns of the GPE⁻ vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE⁻/PAPeV E^rns and vGPE⁻/PhoPeV E^rns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE⁻ vaccine strain. Vaccination at a dose of 10^4.0 TCID₅₀ with either vGPE⁻/PAPeV E^rns or vGPE⁻/PhoPeV E^rns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE⁻/PAPeV E^rns and vGPE⁻/PhoPeV E^rns affirmed their properties, as the vGPE⁻ vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Foot-and-mouth disease (FMD), the most contagious mammalian disease, causes severe economic damage to the livestock industry. Animals infected with FMD virus (FMDV) typically present with vesicles in the mouth, nostrils, and around the breasts and feet. The pathogenicity and infectivity of FMDV in cattle and swine are strain dependent. To investigate the molecular mechanisms underlying these differences, we examined the molecular basis of the pathogenicity of FMDV using two viral strains isolated from two outbreaks of differing severities in Japan. We demonstrated that the two strains have completely different transmissibility in inoculated cattle and virulence in suckling mice. Our study also indicated that VP1 and 3D proteins are independent genetic determinants of this difference. The selectivity of VP1 for host cell receptors and the fidelity of viral RNA during replication were considered important individual factors in the induction of differences in pathogenicity in the host and the severity of outbreaks in the field. These findings provide new insights into the molecular mechanisms underlying the pathogenicity of FMDV.

Because foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 10 ¹ -to 10 ⁴ -fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing 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.