Identification and Comparative Phylogeny of Sheep and Goat Pox Isolates from Jammu, India

Capripoxvirus (CaPV) of small ruminants are most severe of the pox diseases of domestic animals and they have a very important role in agricultural economy. They are included in the list of notifiable diseases of Office International des Epizooties (OIE) (Mirzaie et al., 2015). Sheep pox and goat pox are highly contagious viral diseases of sheep and goat which are caused by the epitheliotropic DNA pox viruses of genus Capripoxvirus, subfamily Chordopoxvirinae and the family Poxviridae.
 
Sheep pox is also known as ovine pox or Laccavalle, caused by virus namely Sheeppoxvirus (SPPV) (Buller et al., 2005), while the other related members of the genus include goat pox virus (GTPV) and lumpy skin disease virus (LSDV) (Van Regenmortel et al., 2000). The causative agents are considered to be very host-specific. Even then, cross-transmission of infection has been reported. Phylogenetic analysis exhibit that members of the Capripoxvirus could be delineated into three distinct clusters of SPPV, GTPV and LSDV based on the P32 genomic sequence (Hosamani et al., 2004).
 
Capripoxviruses are considered to be host-specific and cause outbreaks in a preferred host. However, occasionally some CaPV strains infect both sheep and goats. Instances were recorded when SPPV were found to affect goats in Makhdoom, India (Bhanuprakash et al., 2010) or when GTPV were found to affect both goats and sheep in Samba district of Jammu and Kashmir, India (Ramakrishnan et al., 2016). In view of the limited documentation of sheep and goat pox disease in animals of Jammu region, it was felt necessary to conduct a blanket screening of the small ruminant populations in areas where they are largely reared. Preliminary diagnoses of the disease were to be confirmed by employing molecular techniques with the isolation and characterization of the virus for the circulating strains within the region. The present study therefore was proposed to survey, confirm and characterize the circulating Capripoxvirus strains in Jammu region.

Virus isolates
 
A live attenuated vaccine virus (Raksha-SP) manufactured by Indian Immunologicals (Batch no.01SPPV00317) was used as a reference virus. Viruses obtained from infected tissue or scabs of clinically infected animals were designated putatively as Sheeppoxvirus (SPPV) for isolates obtained from affected sheep and Goatpoxvirus (GTPV) for isolates obtained from affected goats.
 
Collection of clinical samples
 
A survey was carried out for investigating the occurrence of capripox infection in small ruminants from different parts of Jammu, Samba, Kathua, Udhampur, Reasi, Doda, Kishtwar, Rajouri and Poonch districts of Jammu Division (except Ramban district) from June 2017 to November 2018. A total of 47 localities were visited and sheep and goats were investigated for occurrence of the disease. Clinical materials included scabs, skin lesions, or occasional incisional biopsies and lesions from skin from Capripox suspected live or dead animals. Laboratory work was carried out in Veterinary Faculty, Jammu, India.
 
Isolation of putative CaPV (SPPV, GTPV) from clinical samples
 
Clinical scab/skin samples of affected animals were processed for virus isolation. A 10-20% solution of  the   scab  samples in PBS was prepared by mechanical homogenization using a sterilized mortar and pestle. Homogenized samples were centrifuged at 3,000 × g for 15 min. The supernatant was then passed through a 0.45 μm syringe filter (Millipore) and incubated with streptomycin, penicillin antibiotic solution (@ 200 IU of penicillin and 200 µg of streptomycin per ml suspension for 1 hr at 37°C. Processed samples were used for infecting CAM for virus isolation. Inoculation in the artificial air sac route was employed as per Cunningham (1973).
 
Virus isolation in chicken embryonated eggs (CEE)
 
Approximately 10-12 day old embryonated chicken eggs from Government Poultry Hatchery Unit, Belicharana, under Animal Husbandry Department, Jammu were procured for virus inoculation.
 
Detection of CaPV nucleic acid
 
For extraction of viral DNA, commercially available DNeasy Blood and Tissue Kit (Qiagen USA, #69504) was used as per manufacturer’s instructions. The P32 gene encoding a core protein of the virus was targeted using primer sequences (P32 F 5'-TTTCCTGATTTTTCTTACTAT-3'; P32 R 5'-AAATTATATACGTAAATAAC-3') and conditions as previously described by Ireland and Binepal (1998). PCR amplification was performed using 5 µl 10x PCR Buffer, 3 µl MgCl₂ (25 mMol), 0.2 µl dNTPs (10 mMol), 0.3 µl Taq Polymerase (5 U/ µl), 0.5 µl each of primers (10 pmol), 2 µl DNA template and Nuclease free water up to 25 µl in 0.2 ml PCR tubes (Eppendorf, Germany). Cycling conditions were set at 94°C for 5 min for initial denaturation; followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 30 sec and extension at 72°C for 1 min; and final extension at 72°C for 5 min and held at 4°C. The reaction products were separated on a 1.5% w/v agarose gel containing ethidium bromide and visualized in a gel documentation system (BioDoc Analyze, Biometra, Germany).
 
Partial gene sequencing
 
Sequencing of PCR prodects were done commercially for SPPV and GTPV at Sci Genome Lab. Pvt. Ltd. (Kerala, India). Nucleotide sequence corresponding to P32 gene (partial regions) of the Capripox genome was aligned with published sequences obtained from GenBank [using open source BLAST program (National Center for Biotechnology Information, Bethesda MD, http://blast.ncbi.nlm.nih. gov/Blast.cgi for sequence comparison]. For comparison, different sequences comprising all the notified genogroups, isolates from India, neighbouring countries as well as from other countries as well as vaccine strain were used to draw phylogenetic tree. Phylogenetic relationships were determined based on these alignments using clustalW program of MEGA version 5.0 (Tamura et al., 2011) by neighbour joining method. 

Molecular diagnosis and Capripoxvirus confirmation
 
Suspected field samples consisting of skin biopsies and scabs were processed for DNA isolation and subjected to PCR analysis (Table 1). Amplification of Capripoxvirus P32 gene encoding was carried out for- Standard virus (Sheeppox vaccine virus); Tissue lysates prepared from cutaneous scabs containing putative Sheeppoxvirus (SPPV); Tissue lysates prepared from cutaneous scabs containing putative Goatpoxvirus (GTPV); Lysates prepared from putative GTPV inoculated on chicken embryonated egg CAM. Positive samples amplified an expected amplicon size of 192 bp PCR product (Fig 1A, B).
 

 

 
Molecular characterization of different Capripoxviruses
 
After purification of the PCR products, one product from sheep origin (Sheeppoxvirus isolated in Surinsar area of Jammu district) and another of goat origin (Goatpoxvirus isolated in Akhnoor area of Jammu district) were commercially sequenced, annotated and submitted to Genbank, NCBI with published accession numbers (MK358827 and MK358828 respectively).
 
Phylogenetic analysis of different Capripoxviruses
 
Sequence comparison showed that Capripoxvirus isolated from sheep origin grouped under Sheeppox cluster and those isolated from goats grouped under Goatpox cluster (Fig 2). The SPPV strain isolated in the present study was 100% identical with other SPPV strains within India reported from Pune, Tamil Nadu, Bareilly, Akola and even from foreign countries (Tunisia). Interestingly, the SPPV strain was also 100% identical to vaccine strains Srinagar and Rumanian-Fanar (RF). The GTPV closely identical (~98-99%) with other strains from India like Sambalpur and Mizoram and even foreign strains from China, but has some unique signatures of its own. Both the SPPV and GTPV were divergent from Lumpy Skin Disease (LSD) strains. The divergence and relatedness analysis of SPPV and GTPV isolates from Jammu in the present study showed 97.8% identity between the two isolates (Fig 3).
 

 

 
The capripoxviruses have a very large genome and there can be many potential gene targets for diagnosis. The usual choice for using the P32 protein encoding gene is because of its highly conserved nature across all CaPVs and its ability to detect both SPPV and GTPV. The enveloped structural protein contains major immunogenic determinants and therefore has been a preferred target for developing molecular diagnostic tools for detection and differentiation of Capripoxviruses by many workers (Ireland and Binepal, 1998; Heine et al., 1999; Hosamani et al., 2004; Parthiban et al., 2005; Bhanuprakash et al., 2010). It is analogous to P35 protein of Vaccinia virus expressed on the envelope of the mature intracellular virion and may play a role in virus attachment, virulence and virus assembly (Venkatesan et al., 2012). Partial or whole P32 gene can be targeted with specific primers. Even when all CaPVs can be detected, differentiation with GTPV and SPPV can be possible with additional assays, like RFLP analysis (Hosamani et al., 2004; Yan et al., 2010) or by sequence alignment of a part or whole genome of CaPV.

The present results show that there was a high and accurate degree of confirmatory diagnosis based on clinical signs and gross lesions of the disease in both sheep and goats and substantiated by PCR. The application of this method for diagnosing directly from clinical samples is fairly easy and effective.
       
Although the clinical samples and virus isolates in the present study were segregated according to the host species of animal affected, yet a putative designation of GTPV and SPPV was only confirmed after a sequence alignment and phylogenetic construct of the amplified nucleotides. Although earlier reports from some of the areas (Samba district) showed mixed infection in sheep and goats during an outbreak in January 2013 with a GTPV isolate (Ramakrishnan et al., 2016), the present study however demonstrated host specificity of the CaPVs. This has also been substantiated clinically when only either sheep or goats were affected, even when flocks were co-populated with both animal species. Moreover, sequence alignment showed that Capripoxvirus isolated from either animal species clustered under their respective genogroups. However, the possibility of cross transmission cannot be overruled.

Based on the P32 gene, the SPPV isolate shared nucleotide sequence homology with many Indian isolates and some foreign ones. However, homology with vaccine strains makes differentiation between vaccine and field strains difficult. The SPPV nucleotide sequence was 100% identical with both prevalent vaccine strains [Rumanian-Fanar (SPPV-RF) and Srinagar (SPPV-Srinagar) vaccine strains]. Such similarities are also reported for field SPPV isolates elsewhere in the country, either with SPPV-RF vaccine (Hosamani et al., 2004) or SPPV-Ranipet vaccine strain (Parthiban et al., 2005; Rajamuthu et al., 2009). The Rumanian-Fanar (RF) strain licensed and extensively used in India is attenuated from serial passage in primary lamb testes, while SPPV-Srinagar strain has been developed from a local isolate and attenuated in vero cells (Yogisharadhya et al., 2011) and SPPV-Ranipet used in Tamil Nadu has been attenuated in ovine thyroid cells and lamb testes cells (Bhanuprakash et al., 2004). The homology of the circulating Indian SPPV strains favours devising uniform control strategies throughout the country. Since the live attenuated SPPV have been used in enzootic areas, vaccination failure or vaccine induced diseases also cannot be ruled out.
 
The GTPV isolate although sharing nucleotide homology with other Indian isolates (~98-99%), yet there could be subtle differences in nucleotide or amino acid sequences. Earlier reports of such changes have been documented (Santhamani et al., 2014; Ramakrishnan et al., 2016), including presence of an additional aspartic acid at 55^th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus (Hosamani et al., 2004). It is envisaged that any genetic changes or mutations in the viral envelope protein genes, such as the P32 gene which is a major immunogenic protein (Heine et al., 1999), could lead to alterations in the immunogenic properties of the virus. This could lead to vaccination failures when specific GTPV vaccine is not readily available in the country.

From the present study it could be concluded that sheep isolates from Jammu are distinct and found to be identical to many circulating strains in the country and abroad, including some widely prevalent vaccine strains currently in use. It is possible that regular vaccination may help combat the disease in sheep. Goat isolates although similar to the sheep isolate and other circulating strains in the country and elsewhere, still there is subtle divergence seen in the P32 nucleotide sequence and could be unique. Complete P32 gene sequence of the SPPV and GTPV isolates or whole genome sequence is necessary for estimating the relatedness of the field and vaccine strains. Conversely, vaccine challenge studies are warranted for studying the efficacy of prevalent vaccines.