Molecular characterisation of porcine circoviruses in Malaysia and development of a PCV type 3 diagnostic tool

Porcine circoviruses (PCVs) are globally distributed viruses that significantly impact pig populations, with four recognized species: PCV1, PCV2, PCV3, and PCV4. All PCVs share a non-enveloped, icosahedral structure and a single- stranded, circular DNA genome of approximately 2000 nucleotides encoding two main proteins: the replication-associated (Rep) protein and the capsid (Cap) protein. The Cap protein, a major target for host immune responses, is critical for virus detection and pathogenesis studies. PCV1, identified in 1974 as a non-pathogenic contaminant in pig kidney cell cultures, is apathogenic. In contrast, PCV2 emerged in the 1990s as the causative agent of post-weaning multisystemic wasting syndrome (PMWS), a major disease complex that severely affects pig health. The high mutation rate of PCV2, estimated at 10−3 – 10−4 substitutions per site per year, enables rapid evolution and genotype shifts, such as the transition from PCV2b to PCV2d, making it the most extensively studied and epidemiologically significant species. PCV3 and PCV4, identified more recently, are linked to multisystemic diseases, including respiratory, gastrointestinal, and cardiovascular symptoms, with pathogenicity confirmed through infectious clone studies. In Malaysia, PCV2 remains highly prevalent, with an 83.78% detection rate in pig farms and 94.74% in clinically healthy finisher pigs sampled at abattoirs. This study also confirms the presence of PCV3 and PCV4 in Malaysia, marking the first detection of these species in the country’s commercial pig population, with detection rates of 17.02% and 6.67%, respectively. Wild boar samples revealed 100% positivity for PCV2, but PCV3 and PCV4 were not detected, suggesting distinct transmission patterns between domestic and wild pig populations. Phylogenetic analyses revealed a significant genotype shift in PCV2, with most Malaysian strains now classified as PCV2d. Moreover, the findings suggest the introduction of PCV3 and PCV4 into Malaysia via international trade, as Malaysian strains share close phylogenetic relationships with those from neighbouring countries such as Thailand and distant regions like the United States. Interestingly, Malaysian PCV3 strains were phylogenetically related to bat-associated and starling circoviruses, while PCV4 strains were closely linked to bat and mink circoviruses, indicating potential interspecies transmission or common evolutionary origins. This study provides a comprehensive update on the epidemiology of PCVs in Malaysia, emphasizing the endemicity of PCV2 and the emergence of PCV3 and PCV4. The development of advanced diagnostic techniques, including a chromogenic in situ hybridization (ISH) method for PCV3, offers a significant improvement in local diagnostic capabilities. The ISH technique facilitates the identification of PCV3 antigens within infected tissues and aligns well with PCR test results, supporting its utility as a cost-effective diagnostic tool. This advancement complements existing molecular tools like qPCR and aids in addressing knowledge gaps in PCV3 and PCV4 detection and pathogenesis. The findings underscore the need for continued surveillance, improved diagnostics, and biosecurity measures to control PCV transmission and evolution. Applying management strategies developed for PCV2 to PCV3 and PCV4 could mitigate the potential risks posed by these emerging viruses. Further research into the pathogenicity and immune responses associated with PCV3 and PCV4 is crucial for safeguarding the Malaysian swine industry against evolving threats.

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