Capri poxvirus, Sheep pox virus, Polymerase chain reaction (PCR).

Sheep pox viruses are classified within the Poxviridae family, Chordopoxvirinae sub-family, and Capripox virus genus (Buller et al., 2005). These viruses are characterized by their large size (170-260 nm by 300-450 nm), double-stranded DNA, and enveloped structure (Tulman et al., 2002; Biswas et al., 2020). Capripox virus infections in small ruminants represent severe pox diseases affecting domestic animals. In susceptible herds, morbidity rates range from 75-100%, while case fatality rates, contingent upon the virus's virulence, vary between 10-85% during outbreaks. Mortality in older animals can escalate to 90% when capripox is compounded with peste des petits ruminants (PPR). Sheep and goat breeds originating from Europe exhibit high susceptibility to capripox, with mortality potentially reaching 100% (Kitching, 1986; Radostits et al., 2006). 

The Rambouillet sheep breed demonstrates heightened susceptibility to infection compared to Australian cross and American Merino breeds (Mondal et al., 2004). The observed seasonality of sheep pox virus (SGP) may be attributed to the virus's ability to persist for extended periods in wet and cold conditions, its association with the lambing season, or the poor physiological state of flocks in autumn (Bhanuprakash, 2005; Yeruham et al., 2007; Zangana, 2013). The primary vector for SGP virus dissemination is the movement of infected animals (Kitching, 2004; Radostits et al., 2006). The peak shedding of infectious virus and viral DNA in the secretions of infected animals occurs approximately 1-2 weeks post-inoculation, with secretion persisting for an additional 3-6 weeks (Kitching, 1989; Bowden et al., 2008). Stomoxys calcitrans, once infected, may transmit the pox virus to susceptible goats (Bhanuprakash et al., 2006). The presence of antibodies against a virus in animal species indicates susceptibility; however, animals with such antibodies may not develop the infection or transmit the virus (Tuppurainen and Oura 2012).

There is limited information regarding the epidemiology and economic impact of sheep pox (SP) in dairy sheep flocks. This disease results in significant economic losses due to morbidity, mortality, decreased meat and milk production, abortion, and the depreciation of wool and skin quality, as well as trade restrictions (Yeruham et al., 2007). Economic analyses have identified several variables, such as the number of adult animals affected, the duration of illness, and flock size, as significantly influencing the economic losses associated with sheep pox (Garner et al., 2000; Senthilkumar and Thirunavukkarasu 2010). The incubation period of sheep and goat pox (SGP) ranges from 4 to 14 days; however, the OIE has recorded a maximum incubation period of 21 days (OIE, 2010). Typically, the initial symptoms of the disease include swelling of the nostrils, followed by thick nasal discharges and watery ocular discharges. Infected animals exhibit elevated body temperatures (41-42 C), and keratitis may develop (Daoud, 1997). 

In the malignant form of sheep pox, symptoms such as high fever, marked depression, ocular and nasal discharges, and pock lesions on hairless areas of the skin, as well as on the mucosa of the respiratory, digestive, and urogenital tracts, have been observed. Pock lesions on the skin initially present as papules, which then progress to nodular, pustular, and finally scab stages. Pock lesions have also been observed in the heart muscles, although this manifestation of the disease is rare. In the benign form, more common in adult animals, only skin lesions occur, particularly under the tail, with no systemic reaction, and animals typically recover within 3-4 weeks. Abortion and secondary pneumonia have been reported as complications of sheep pox (Radostits et al., 2006). Animals that recover from sheep pox infection acquire lifelong immunity. The virus persists through continuous transmission from infected to susceptible animals, necessitating a minimum size of the susceptible population (Kitching, 2004). The size of this population is influenced by the virus strain, host population susceptibility, and the number of infected animals (Panchanathan et al., 2008; Sadri and Fallahi 2010). 

Active mass vaccination against sheep pox can induce strong herd immunity, effectively controlling the disease. A single vaccination has been reported to provide lifelong strong immunity (Bhanuprakash et al., 2011). At least two different vaccines containing isolates of the SP virus are available to protect small ruminants against sheep pox (Carn, 1993; Rao and Bandyopadhyay 2000; Abu-Elzein et al., 2003; Radostits et al., 2006; OIE, 2013; Iran Veterinary Organization, 2014). The Kenyan vaccine strain (O-240), not considered an SP virus, has been associated with clinical disease when used as a vaccine (Tuppurainen et al., 2014). In India, the occurrence of sheep pox in both sheep and goat flocks within the same geographical location has been reported (Bhanuprakash et al., 2010).

Capripox viruses exhibit a high degree of host specificity, with most isolates predominantly affecting either sheep or goats (Bhanuprakash et al., 2010). According to Kenubih et al. (2021), GTPV has been accountable for the occurrence of CaPV diseases in small ruminants. Due to the close antigenic and virulence relationship between sheep pox virus (SPV) and goat pox virus (GPV), serological assays are unable to differentiate between the two (Balinsky et al., 2008). However, phylogenetically distinct serotypes can be identified through molecular characterization targeting specific Capripox virus genes, such as RPO30, GPCR, and P32 (Bhanuprakash et al., 2006; Yan et al., 2012; Biswas et al., 2020).  Some sheep strains cause mild disease in goats and severe disease in sheep, whereas virulent goat strains can infect sheep (Bhanuprakash et al., 2010; He et al., 2020). 

The prompt detection of SPV is crucial for effective control and mitigation of the significant economic losses associated with outbreaks (Mangana-Vougiouka et al., 2000). Diagnosis typically relies on clinical signs followed by laboratory confirmation, which primarily involves virus isolation and electron microscopy (EM) (Oguzoglu et al., 2006). Conventional seromonitoring techniques, including immunofluorescence, immunoprecipitation, virus neutralization, and ELISA, are widely employed for routine diagnosis. Previously, a polymerase chain reaction (PCR) method based on fusion and attachment protein genes has been reported to offer greater sensitivity than antigen trapping ELISA (Ireland and Binepal 1998). In the face of adversity, inactivated Capripox vaccines are safe and efficacious against CaPV (Wolff et al., 2021; Es-sadeqy et al., 2021). 

Sample Collection: Sheep pox virus (SPV) infection is characterized by symptoms such as pyrexia, oculo-nasal discharge, and pock lesions that range from erythema to scabs on the body, with occasional pulmonary nodules (Rao and Bandyopadhyay 2000), as illustrated in Photos (1, 2, and 3). In this study, sixty-two clinical samples, including skin nodules, lung tissues, and crusted scabs, were collected from unvaccinated sheep exhibiting SPV symptoms. The samples were transported to the laboratory in a transport medium (PBS) containing antibiotics, maintained under chilled conditions, and stored at -40ºC until further analysis. 

Polymerase Chain Reaction: DNA Extraction: DNA extraction from the samples was conducted using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH), with modifications to the manufacturer's protocol. Specifically, 200 μl of the sample suspension was incubated with 10 μl of proteinase K and 200 μl of lysis buffer at 56°C for 10 minutes. Following incubation, 200 μl of 100% ethanol was added to the lysate. 

The sample was subsequently washed and centrifuged according to the manufacturer's instructions. Nucleic acid was eluted with 200 μl of elution buffer provided in the kit (Yousif et al., 2010). The primers used are listed in Table (1), and the cycling conditions for the ORF103 and RPO30 genes are depicted in Table 2. Primers were utilized in a 25-μl reaction containing 10 μl of PCR Master Mix, 1 μl of each primer at 20 pmol concentrations, 3 μl of water, and 5 μl of DNA template. The reaction was performed in a thermal cycler (Lamien et al., 2011). 

The PCR products were separated by electrophoresis on a 1.5% agarose gel in 1x TBE buffer at room temperature, using a gradient of 5V/cm. For gel analysis, 5 μl of the products was loaded into each gel slot. A 100 bp DNA Ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed using a gel documentation system (Bio-Rad), and the data were analyzed using computer software.

The application of conventional polymerase chain reaction (PCR) for nucleic acid detection yielded positive results across all prepared samples, as evidenced by the amplification of characteristic 151 base pair (bp) fragments of sheep pox viral DNA, clearly depicted in Fig. 1. The agarose gel, stained with Ethidium Bromide, exhibited prominent bands, thereby visually confirming the presence of viral DNA in the chorioallantoic membrane (CAM) suspensions. This initial screening method demonstrated efficacy in identifying potential sheep pox virus infections within the sample set.  

To further substantiate the findings, an additional amplification step targeting a 570 bp fragment from the ORF 103 gene was conducted on three selected positive samples (Biswas et al., 2020). This secondary confirmation enhanced the reliability of the results. The study's overall outcome indicated a significant prevalence of sheep pox virus, with 46 out of 62 samples (74.19%) testing positive based on the PCR analysis. The presence of strong bands in the Ethidium Bromide-stained agarose gel, as illustrated in Figure 2, provided clear visual evidence of viral DNA amplification, reinforcing the PCR results and underscoring the effectiveness of this diagnostic approach in detecting sheep pox virus infections.

DISCUSSION 

Sheep pox, induced by the sheep pox virus (SPV), represents a severe and highly contagious disease that poses a substantial threat to global sheep populations. The virus is classified within the Capripoxvirus genus, which also encompasses the goat pox virus (GPV) and the lumpy skin disease virus. Clinically, the disease is characterized by a spectrum of symptoms, including elevated fever, respiratory distress, and the development of distinctive pox lesions on the skin and internal organs. These manifestations can result in diminished productivity, economic losses, and, in severe instances, mortality among affected flocks (Garner et al., 2000; Bhanuprakash et al., 2006). The effective management of sheep pox outbreaks necessitates a comprehensive approach, integrating rapid and precise diagnostic methods with strategic vaccination initiatives. While traditional diagnostic techniques such as virus isolation and electron microscopy retain their value, molecular methods like PCR have significantly enhanced the speed and sensitivity of detection. Nevertheless, the close antigenic similarity between SPV and GPV complicates serological differentiation, necessitating the development of more specific diagnostic tools (Balinsky et al., 2008). This highlights the importance of ongoing research to advance diagnostic capabilities and improve vaccine efficacy, ultimately contributing to the enhanced control and potential eradication of sheep pox in endemic regions (Bhanuprakash et al., 2011). 

Alternative molecular methods for identifying and distinguishing sheep pox virus (SPV) from goat pox virus (GPV) have been developed to augment diagnostic capabilities. These methods include restriction endonuclease analysis (REA), whole genome sequencing, P32 gene-based PCR-RFLP, PCR targeting the RPO30 gene, and GPCR gene-based quantitative PCR (Hosamani et al., 2004; Lamien et al., 2011). Among these techniques, PCR targeting the RPO30 gene has demonstrated particular efficacy in differentiating between SPV and GPV (Santhamani et al., 2013). In this study, SPV was successfully identified in samples isolated and cultured in embryonated chicken eggs using the chorioallantoic membrane (CAM) route. The presence of SPV was confirmed through PCR analysis using primers specific to the RPO30 gene, resulting in the amplification of 151 bp fragments. These findings were further validated through gel electrophoresis (Fig. 1), consistent with previous research by Lamien et al. (2011); Yan et al. (2012) on Capripoxvirus (CaPV) species identification. 

To further reinforce the identification of SPV, the study also targeted the ORF 103 gene, successfully amplifying a 570 bp fragment from samples suspected of sheep pox infection (Fig. 2). This multi-gene approach enhances the reliability of SPV detection and differentiation from other closely related poxviruses. The combination of virus isolation techniques, such as culturing in embryonated chicken eggs, with molecular methods like PCR and gel electrophoresis, provides a robust framework for accurate diagnosis and characterization of SPV. These findings contribute to the expanding body of knowledge on poxvirus diagnostics and underscore the importance of employing multiple molecular targets for comprehensive virus identification.

The successful isolation of a field strain of the sheep pox virus from infected sheep constitutes a significant advancement in the understanding and management of this infectious disease. Sheep pox, caused by the sheep pox virus (SPPV), is a highly contagious and potentially lethal disease affecting sheep populations globally. The isolation of the virus from infected specimens provides researchers with valuable live samples for further investigation, facilitating a more precise characterization of the pathogen and its behaviour within host organisms. The study's application of PCR assays targeting the RPO30 and ORF 103 genes, in conjunction with sequencing techniques, presents a robust method for identifying sheep pox virus infections. This approach not only enhances diagnostic capabilities but also lays the groundwork for the development of more effective vaccination strategies. By accurately identifying the virus and its genetic composition, researchers can design vaccines that specifically target the most pertinent viral antigens, potentially enhancing immune responses and overall protection against sheep pox. Moreover, this methodology may contribute to epidemiological studies, enabling improved tracking of virus strains and their geographic distribution, which is essential for implementing targeted control measures and preventing outbreaks in susceptible sheep populations.

Table 1: Primers sequences, target genes, amplicon sizes.

Table 2: Cycling conditions – PCR (ORF103 & RPO30 genes).

Fig. 1. Amplification of RPO30 gene of sheep pox virus by PCR. M: molecular marker. Lane (1): positive control Lane (2-5): positive PCR products from sheep pox virus isolate (CAM). Lanes (6): negative control

Fig. 2. Amplification of ORF 103 gene by PCR. M - DNA marker, lane (1): positive control; lane (2 - 5): positive isolate; lane (6): Negative control.

Expanding on the epidemiological perspective of sheep pox virus, further investigation into the sequence analysis of immunomodulatory protein genes is crucial for developing effective control strategies. These genes play a vital role in the virus's ability to evade host immune responses and establish infection. By conducting comprehensive sequence analyses across different strains and isolates of sheeppox virus, researchers can identify conserved regions and potential targets for vaccine development or antiviral therapies. Moreover, understanding the genetic diversity and evolution of these immunomodulatory proteins can provide insights into the virus's adaptation mechanisms and potential cross-species transmission. This knowledge is essential for predicting and mitigating future outbreaks, as well as designing diagnostic tools with improved sensitivity and specificity. Additionally, comparative genomic studies with related poxviruses may reveal common immunomodulatory strategies, facilitating the development of broad-spectrum control measures that could be effective against multiple poxvirus infections in sheep and potentially other livestock species.

Manickam R., Puvarajan B., Balasubramaniam A., Balakrishnan S. and Selvaraj J.  (2024). Molecular Detection of Sheep Pox Virus and its Differentiation from Goat Pox Virus. Biological Forum – An International Journal, 16(11): 168-173.