Molecular characterization of Indian sheeppox and goatpox viruses based on RPO30 and GPCR genes

Abstract

Sheeppox and goatpox are economically important diseases of small ruminants caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively. Although SPPV and GTPV have host preference, some strains may infect both sheep and goats. As capripox viruses (SPPV, GTPV and LSDV) are antigenically related but genetically distinct, their differentiation requires analysis at molecular level. In the present study, RPO30 and GPCR genes of eight Indian SPPV and GTPV isolates were PCR amplified, cloned and sequences are genetically and phylogenetically analyzed. The RPO30 gene of SPPV and GTPV had lineage-specific signatures, and deletion of 21-nucleotide exclusively present in SPPV. Similarly, GPCR gene also had lineage-specific signatures for SPPV and GTPV. Phylogenetic analysis of capripox viruses based on RPO30 and GPCR genes revealed three distinct lineage-specific clusters as per their host origin. Our study supports that both RPO30 and GPCR genes could be used for differentiation of SPPV and GTPV as well as for molecular epidemiological studies. The study also highlights the distinct lineage specificities of the Indian SPPV and GTPV isolates including vaccine strains.

Introduction

The Capripoxvirus genus, one of the ten members (http://www.ictvonline.org/virusTaxonomy.asp?version=2013) of the subfamily Chordopoxvirinae, family Poxviridae comprises three important pathogens of ungulates, i.e., sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV; not reported in India) [1], which infects sheep, goats and cattle, respectively. Capripox virions have an ovoid shape with an average size of 294 × 273 nm. Their genome is linear double-stranded DNA of ~151 kb, contains 156 putative genes with high AT content of 73–75 % and shares 96 % nucleotide identity [2, 3]. Among the 156 ORFs, the essential genes of replication, structure and assembly are located in central conserved ORFs 024 to 123 and virulence and host range functions are in terminal variable ORFs 01 to 023 and 124 to 156 [3, 4].

Capripoxviruses (CaPVs) are currently classified based on the host species from which the virus was originally isolated. Subsequently, some SPPV and GTPV isolates were found to be pathogenic to both sheep and goats [5–9]. As both SPPV and GTPV produce similar clinical signs, and antigenically related, they are indistinguishable by serological tests. For many years, CaPV genotyping has been based on the electrophoretic patterns of viral genome isolates following digestion with restriction enzymes [10]. This method is time-consuming and requires large amount of viral material, therefore cannot be applied in a routine basis. Recent molecular-based studies have shown that CaPVs are phylogenetically and genetically distinct giving a basis for the molecular differentiation of CaPVs [11–13]. RPO30, GPCR and P32 gene sequence analysis were used for differentiation of SPPV and GTPV [11–14].

RPO30 gene (ORF 036), homologue of Vaccinia virus E4L gene, encodes the RNA polymerase 30 kDa subunit plays a role in replication [14]. GPCR gene (ORF 011), a host range gene encodes the G-protein-coupled chemokine receptor, is a membrane-bound protein involved in host immunomodulation [13]. RPO30, GPCR and P32 genes possess the specific signatures for SPPV and GTPV at both nucleotide and amino acid sequences level; thus, they were used for the differentiation of SPPV and GTPV [11–14]. In India, both SPP and GTP occur often and cause high annual losses (~INR 1250 million) due to their enzootic nature [15]. Losses in farmer’s income may reach 30–43 %, and it will take an average of 6 years for a flock or herd to return to normal production levels [16]. Regular outbreaks of sheeppox [17–20] and goatpox [21, 22] were reported throughout the country despite of using homologous live-attenuated vaccines [23]. Using vaccine strain (SPPV or GTPV) derived from an isolate which has equal pathogenicity to both sheep and goats could give complete protection against sheeppox and goatpox [24]. However, only limited reports are available on molecular characterization of SPPV and GTPV for their exact lineage and host specificity. The present study describes the characterization of Indian SPPV and GTPV isolates which include vaccine and cell culture-adapted viruses.

Materials and methods

Viruses

A total of eight viruses (4 SPPV and 4 GTPV) were used in this study. SPPV vaccine Roumanian Fanar p35 was procured in freeze-dried form from Division of Biological Products, Indian Veterinary Research Institute (IVRI), Izatnagar, India. Three SPPV including two vaccine strains (Ranipet p54, Srinagar p39) and one isolate (Srinagar p4); four GTPV including three field isolates (Mukteswar p3, Sambalpur p8, Akola p1) and one vaccine strain (Uttarkashi p60) are maintained in the Virus Repository of Pox virus laboratory, Division of Virology, IVRI, Mukteswar, India. All the above eight viruses were propagated once in Vero cells using Eagle’s minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 % horse serum (Invitrogen, Grand Island, NY, USA). The infected cells were harvested on 7th dpi (80 % of cells showed cytopathic effect) and stored at −80 °C.

DNA extraction

The DNA extraction from cell culture lysate was carried out using FNES (fast, non-enzymatic and, simple) method [25] with two additional steps, viz., precipitation of DNA with 100 % isopropanol and washing with 70 % ethanol [26]. The DNA pellet was resuspended in 30 µL of nuclease-free water and stored at −20 °C. All the DNA samples were checked for identity using capripoxvirus-specific RPO30 gene-based PCR [14].

Amplification of RPO30 and GPCR genes

Full-length RPO30 and GPCR genes were amplified from the extracted DNA using the primer pairs, RPO30-1 (5′-CTCTGTTCCAAACTAAATCAT-3′) and RPO30-2 (5′-TTTTTGTATTACCAATTTCTG-3′), and GPCR-1 (5′-TTTATCAGCACTAGGTCATTATCT-3′) and GPCR-2 (5′-TATCACTCCCTTCCATTTTTAT-3′) [27], respectively (IDT, USA). The PCR was carried out in a 100-μL reaction mixture in containing 50 μL Maxima Hot Start Green PCR Master Mix (Thermo Scientific, USA), 2 μL of each primer (10 pmol/μL), 2 μL of extracted DNA and 42 μL of nuclease-free water with an initial denaturation at 95 °C for 4 min and 35 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C (RPO30 gene)/59 °C (GPCR gene) for 1 min and extension at 72 °C for 1 min, with a final extension of 10 min. The expected amplification was confirmed by electrophoresing the PCR products along with 100 bp plus DNA ladder (Fermentas, USA) in 1.5 % agarose gel containing ethidium bromide (0.05 µg/mL) at 100 V for 60 min and visualized in transilluminator.

Cloning and sequencing of RPO30 and GPCR genes

The PCR products were purified using AuPreP PCR purification kit (Life Technologies India, New Delhi) and cloned into pTZ57R/T vector using InsTAclone PCR Cloning Kit (Thermo Scientific, USA) and commercially sequenced (SciGenome, Cochin, India). The GenBank accession numbers of the nucleotide sequences are presented in Table 1.

Table 1 Details of nucleotide sequences of RPO30 and GPCR genes of capripox viruses used in the analysis

Genetic and phylogenetic analysis of RPO30 and GPCR genes

RPO30 and GPCR genes nucleotide and their deduced amino acid sequences of SPPV and GTPV were analyzed by comparing with respective reference sequences obtained from the GenBank (Table 1). Multiple sequence alignment and pairwise distance calculations were carried out using ClustalW algorithm in MegAlign (Lasergene v6; DNAStar, USA). The phylogenetic tree was constructed using neighbor-joining algorithm with Kimura-2 parameters correction and 1000 bootstrap replications with the MEGA 5 [28].

Results

Genetic and phylogenetic analysis of SPPV and GTPV based on RPO30 gene

The cloned amplicons were sequenced and complete ORF length of 606 bp, and 585 bp were obtained for GTPV and SPPV isolates, respectively. In both nucleotide (nt) and deduced amino acid (aa) sequence-based phylogenetic analysis, the Indian SPPV and GTPV isolates formed two distinct clusters as per their host origin. Although there was a lower bootstrap value, two subgroups were observed within GTPV lineage. All the four GTPV isolates used in the present study clustered with GTPV India/83, GTPV Bangladesh/86, GTPV Oman/84 and GTPV GS-V1 (Fig. 1). In case of SPPV, all the isolates including the currently studied isolates formed only a single cluster (Fig. 1).

Fig. 1

Phylogenetic analysis of SPPV and GTPV based on RPO30 gene

Multiple nucleotide sequence alignment of RPO30 gene of SPPV revealed a deletion of 21-nts (from ORF position 13–33) compared to GTPV sequences. The lineage-specific aa signatures for SPPV and GTPV were observed at positions 92, 96 and 135 (Table 2). Further, unique aa signatures were observed for few isolates. SPPV Roumanian Fanar, SPPV Ranipet and SPPV Morocco vaccine strains showed lysine (K) instead of asparagine (N) at position 190 (N190K). Similarly, SPPV Srinagar p40 and p5, and GTPV SA 93 had glutamate (E) in place of aspartate (D) at position 43 (D43E). Isoleucine (I) was present at position 6 in Indian GTPV isolates, GTPV Bangladesh/86 and GTPV Oman/84, whereas tryptophan (W) was observed in other foreign GTPV isolates. All the four Indian GTPV isolates used in the present study showed 100 % identity at both nt and aa levels with each other as well as with GTPV India/83, GTPV Bangladesh/86, GTPV Oman/84 and GTPV GS-V1. However, they shared 99.3–99.8 % nt and 98.5–99 % aa identity with other foreign isolates. SPPV Roumanian Fanar and Ranipet shared 100 % nt identity with each other and shared 99.5 % nt identity with SPPV Srinagar p40 and Srinagar p5. Indian SPPV isolates had 99.3–99.5 % nt and 99–99.5 % aa identity with foreign SPPV isolates. Indian GTPV and SPPV shared 93.4–93.6 % nt 94.6 % aa identity between them and shared 98 and 97.4 % aa identity, respectively, with LSDV.

Table 2 SPPV and GTPV lineage-specific amino acid signatures observed in the RPO30 and GPCR genes

Genetic and phylogenetic analysis of SPPV and GTPV based on GPCR gene

Complete ORF (1,125 bp in both GTPV and SPPV) sequence was obtained for all the SPPV and GTPV isolates. In both nt and their deduced aa sequence analysis, all the SPPV and GTPV isolates formed distinct clusters according to their host origin. Unlike RPO30 analysis, GPCR-based phylogeny revealed two distinct subgroups with strong bootstrap value (99 %) in GTPV lineage, i.e., subgroup 1 and subgroup 2. All the four GTPV isolates used in this study clustered in subgroup 1 along with GTPV Bangladesh/86, GTPV Oman/84, SPPV Oman/84 and GTPV GS-V1. All the SPPV clustered under single group (Fig. 2).

Fig. 2

Phylogenetic analysis of SPPV and GTPV based on GPCR gene

GTPV isolates used in the present study shared 99.8–99.9 % nt and 99.7–100 % aa identity among themselves as well as with GTPV India/83, GTPV Bangladesh/86, GTPV GS-V1 and SPPV Oman/84. They had 97.7–99.8 % nt and 96.8–97.3 % aa identity with other foreign GTPV isolates.

Indian SPPV isolates had 99.7–99.8 % nt and 99.2–99.7 % aa identity among them and had 99.5–99.9 % nt and 96–99.7 % aa identity with foreign SPPV isolates. SPPV Srinagar p40 and SPPV Srinagar p5 had 100 % identity between them as well as with GTPV SA/93. Indian GTPV and SPPV shared 94.8–95.4 % nt and 93.1–94.1 % aa identity, whereas they had 94.7–95.2 % nt and 91.8–92.3 % aa identity with LSDV.

In multiple sequence alignment of GPCR analysis, all the eight Indian capripox virus (SPPV and GTPV) isolates revealed the absence of 21-nucleotide (7-aa) at the 5′ end of GPCR gene compared to some foreign GTPV isolates which possess additional 21-nts at this position. It was observed that 23 lineage-specific aa signatures that can be used as markers for the differentiation of SPPV and GTPV. Among 23, only 3 aa (at position 34, 99 and 199) are lineage specific, i.e., aa signatures present either in SPPV or GTPV or LSDV (Table 2), whereas remaining 20 aa signatures are common for either two of the three capripox viral species. SPPV Roumanian Fanar and SPPV Morocco possessed serine (S) at position 92 instead of phenylalanine (F92S). SPPV Ranipet substituted with cysteine (C) at position 117 for arginine (R117C). Also GTPV Sambalpur had histidine (H) at position 205 instead of tyrosine (Y205H). Overall, GPCR gene is found to be highly conserved within the lineages.

Discussion

Although both sheeppox and goatpox diseases are endemic in India, reports on molecular characterization of capripoxviruses for differentiation of species are scanty [11]. Due to the large genome size of the poxvirus, multiple genes or whole genome analysis is necessary to supplement the available molecular data [27]. The present study describes the molecular characterization of Indian SPPV and GTPV isolates based on RPO30 and GPCR genes. These genes are carrying specific molecular signatures for SPPV and GTPV and can be used for differentiating these viral species/lineages [12–14].

RPO30 gene of Indian isolates of SPPV and GTPV had 585 and 606 bp, respectively. The virus species/lineage-specific markers such as 21-nts deletion in SPPV at the 5′ end and other signatures were observed. Similar results were already reported for foreign isolates [14]. It was reported that the length of GPCR gene of SPPV is 1,125 bp, whereas GTPV is either 1,125 or 1,146 bp [13]. Interestingly, the GPCR gene of all the eight Indian SPPV and GTPV isolates had 1,125 bp.

Although several genes are reported as host range genes, studies on attenuation markers are limited [3]. Presence of N190K in RPO30 gene of the Indian SPPV vaccine strains (SPPV Roumanian Fanar and SPPV Ranipet) and SPPV Morocco vaccine strain, indicating that this signature molecule may have a role in the attenuation process. Similarly, SPPV Roumanian Fanar and SPPV Morocco had F92S as specific signature in GPCR gene. In both RPO30 and GPCR analysis, SPPV Srinagar P5 (virulent challenge virus) and SPPV Srinagar P40 (vaccine virus) had 100 % identity. The above results indicate that mutations in some other genes might have played in a role of attenuation. Le Goff et al. [13], also opined that the GPCR gene is not involved in attenuation. Both RPO30- and GPCR-based phylogenetic analysis revealed the formation of three distinct lineages, viz., SPPV, GTPV and LSDV. Further, GTPV lineage formed two subgroups. These results are corroborated with other group of researchers who reported three distinct lineages and subgroups in GTPV [13, 14, 27]. Zhou et al. [27] observed three subgroups within the GTPV based on P32 gene. The P32 gene analysis of Indian capripoxviruses also shows similar results [11]. SPPV Srinagar is closely related to GTPV SA/93. However, GTPV SA/93 and SPPV Oman/84 were named based on host origin, and later, they were reclassified as SPPV and GTPV, respectively, based on RPO30 and GPCR gene analysis [12–14].

In conclusion, both RPO30 and GPCR genes possess lineage-specific signatures for sheeppox and goatpox viruses and could be used for differentiation of SPPV and GTPV as well as for molecular epidemiological studies. Our study confirms that the currently in use Indian sheeppox and goatpox vaccines and challenge strains are lineage specific. Although RPO30 and GPCR genes have been used for the differentiation of SPPV and GTPV, it is not clear whether they play a role in host specificity or not. The RPO30 and GPCR gene sequence analysis of SPPV/GTPV infecting both sheep and goats may shed light on host-specific markers as well as single-strain broad-spectrum vaccine development.