All clinical samples were found positive for LSD indicating the disease spectrum among the cattle population. The PCR results were fully correlated to the field-based diagnosis of typical clinical symptoms of LSD. The PCR technique detected LSDV in skin nodules swab samples suggesting the best source for virus detection compared to other sources such as blood, semen, and milk as they contain more viral particles due to the tissue tropism nature of LSDV (Tuppurainen et al., 2005; Babiuk et al., 2008). The detection of the virus in skin wounds also suggested that the virus could be transmitted among the cattle population via direct contact with the virus shed from skin wounds or body excretion which has been observed recently in a previous study (Aleksandr et al., 2020). In the current study, the phylogenetic analysis was conducted utilizing the partial GPCR (1030bp), p32 (174bp), and complete RPO30 (606) genes of LSDV strain originating from different locations across two different provinces. Topology of the partial GPCR gene revealed that LSDV strains originating from outbreaks in Pakistan noticeably clustered together with LSDV strains reported from Russia, Egypt, India, and Turkey (Fig. 2A). The PASC analysis of the GPCR gene showed that under-studied LSDV strains had 99.6%-99.8% genomic identities with LSDV strains reported from Russia, Turkey, Egypt, and India. On the other hand, these LSDV strains had 99.1%-99.5% genomic identities with LSDV strains reported from Africa and 98.5% genomic identity with vaccine strains. Topology of the complete RPO30 gene (606 bp) revealed that LSDV strains originating from outbreaks in Pakistan clustered together with LSDV strains reported from Middle East, African, and Asian countries (Fig. 2B). The PASC analysis revealed that these LSDV strains had 99.8%-99.9% genomic identities with LSDV strains reported from India and Bangladesh, 99.5% with LSDV strains reported from Russia, 99.2% with LSDV strains reported from Thailand, 99% with LSDV strains reported from Africa, 98.8% with vaccine strains. These substantial analyses revealed that LSDV strains were however most closely related to strains from a wide geography and therefore, suggesting that the same LSDV strains are responsible for outbreaks across borders (Ochwo et al., 2020; Wang et al., 2022). The PASC analysis revealed that all LSDV strains originating from Pakistan had 100% genomic homology suggesting the circulation of identical or similar LSDV strains in the whole country and could be responsible for more LSDV outbreaks in the country as evidenced by previous studies exploring partial p32 gene (Sudhakar et al., 2020; Ma et al., 2021; Selim et al., 2021).
Topology of the partial p32 gene (174 bp) revealed that LSDV strains originating from outbreaks in Pakistan noticeably clustered together with LSDV strains reported from Middle East and Asian countries including Egypt, Kingdom of Saudi Arabia, India, and China (Fig. 2C). Such findings are not unusual due to similar outcomes are now observed in various epidemiological investigations conducted in wide geography (Şevik and Doğan, 2017; Aleksandr et al., 2020; Ma et al., 2021; Kumar et al., 2021; Maw et al., 2022). The PASC analysis revealed that these LSDV strains had 99.6%-99.9% genomic identities with LSDV strains reported from Asian countries including India, China, Bangladesh, and Vietnam while 98.8% genomic identity with vaccine strains. On the other hand, under-studied LSDV strains had 98% genomic homology with vaccine strains. The highest genomic similarity (98%-99.9%) between LSDV strains reported from different geography and vaccine strains highlighted the previous theory that all capripox viruses are genetically related and originated from one ancestral lineage (Tulman et al., 2001). Noteworthy is the fact that currently available vaccines containing vaccine strains may provide adequate protection against LSDV infection as previously observed (Klement et al., 2020; Wolff et al., 2020) and suggested a vaccination campaign to control LSDV outbreaks, particularly in highly risky regions nearby areas where outbreaks are now confirmed in Pakistan (Fig. 1).
Collectively, the genomic analysis revealed that under-studied LSDV strains have shared the highest genomic homology with strains reported from India, China, and Bangladesh, suggesting the circulation of similar strains across the borders (Hasib et al., 2021; Sudhakar et al., 2022; Wang et al., 2022). There are possible chances that affected animals may move across borders in search of pasture and water and may introduce LSDV endemic regions to non-endemic regions (Sprygin et al., 2019). Before the outbreaks of LSD in Pakistan, the disease was reported by OIE in India and therefore as it was speculated in our previous work (Ul-Rahman et al., 2022), LSD could have entered Pakistan by uncontrolled movement of infected animals from India. Owing to the potential role of vectors, including flies and ticks, in the dissemination of the virus, there is a possible chance that vector mitigation introduced LSDV in Pakistan from India (Tuppurainen et al., 2011; Sohier et al., 2019). While comparing phylogenetic patterns constructed using different genes, the topology of p32 gene clustered the field strains precisely far away from vaccine strains along with distinct geographical patterns indicating the possibility of using p32 gene as a candidate for differentiating field prevailing and vaccine strains in a wide geography. The p32 gene is homologous to the P35 protein encoded by vaccinia virus H3L and corresponds to an envelope protein for the attachment of intracellular viral particles (Tulman et al., 2001). It is a highly conserved region and has usually been used as a reliable diagnostic tool for determining the origin and illustrating genetic variations between LSDV strains (Mafirakureva et al., 2017; Mansour, 2017; Rashid et al., 2017).
The reasons for the origin and spread of LSD to Pakistan are unknown but it may be due to livestock movement across international borders or may be due to vector movement from neighbouring countries. Recently LSD outbreaks have been reported in neighbouring countries including India, Iran, and China (Sudhakar et al., 2020; Hedayati and Varshovi, 2021; Lu et al., 2021). Pakistan has not experienced LSDV infection previously and currently has a large number of cases among the cattle population of more than nine million presently and the risk of disease spread to 41 million cattle across Pakistan. The findings of the current study indicate that, despite the weak regulations governing vaccine acquisition in Pakistan, the current outbreaks are most probably caused by wild type. Currently, the epidemic status and affected regions of LSD across Pakistan are not fully investigated. Therefore, it is crucial to investigate outbreaks in wide geography considering all provinces across the country with the possibility of the significant complete gene or genome sequencing for the illustration of molecular epidemiology of LSDV in the country for the establishment of effective disease control strategies and implementation of a well-designed vaccination program against the virus.