Molecular epidemiology of swinepox viruses circulating in India

Abstract Swinepox is a sporadic virus disease of domestic and wild pigs that mainly occurs during the rainy season. Though the disease is known for a century, research on swinepox especially genetic characterization is scanty. Self-limiting nature of the disease, the non-availability of specific diagnostics as well as the resemblance of clinical signs with other pathogens are some of the issues in the slow progress in swinepox-related research. Recent whole genome sequencing data from the USA, India, and Germany enhanced our understanding of the biology of swinepox virus (SWPV). The objective of the present study is to investigate the molecular epidemiology of two swinepox outbreaks that occurred in 2015 and 2016 one each in Uttar Pradesh, and the Haryana states of India. The appearance of clinical signs in different swine breeds was recorded. The scab samples from infected pigs were collected, DNA extracted, host range genes of SWPV were PCR amplified, sequenced and analyzed for genetic and phylogenetic characterization. Desi (nondescript breed), Yorkshire White pigs, and Landrace cross were found to be infected with SWPV. Host range genes of SWPV analyzed from clinical samples showed very high nucleotide identity with each other. Phylogenetic analyses revealed that SWPVs circulating in India are distinct (Indian lineage) from the SWPV of the USA, Germany, and Russia (European-North American lineage). Our study affirms the existence of two distinct lineages of SWPV globally with differences in clinical lesions between breeds.


Introduction
Swinepox virus (SWPV), the causative agent of swinepox disease, has been classified as the only member of the genus Suipoxvirus in the subfamily Chordopoxvirinae of the Poxviridae family (Delhon et al. 2007). Swinepox has been reported in North America, South America, Europe, and India (Ramakrishnan and Ashokkumar 2019) with outbreaks occurring mainly during the rainy season. The primary cause of the spread of the disease is inadequate sanitary conditions where pigs are raised. In addition to direct contact and congenital infection, it is believed that the virus could be transmitted mechanically by pig lice (Haematopinus suis) and domestic flies (Musca domestica) (Borst et al. 1990;Thibault et al. 1998;Delhon et al. 2007;Mittal et al. 2011;Kaiser et al. 2021). SWPV infection is associated with high morbidity in piglets of 3-4 months old and pigs are the only hosts and reservoirs of this pathogen. SWPV-elicited disease in adults is less pathogenic, self-limiting, and of limited economic impact. Several outbreaks have been reported in Assam, and Haryana states of India (Mittal et al. 2011;Jindal et al. 2015;Riyesh et al. 2016;Mech et al. 2018;Aasdev et al. 2021). Recently, swinepox outbreaks were reported in both domestic and wild pigs in Germany (Kaiser et al. 2021). The condition develops mainly as a skin pustular lesion, including hydropic degeneration of skin stratum spinosum. Swinepox diagnosis can be achieved by histopathology (Cheville 1966a), electron microscopy (Teppema and De Boer 1975;Mittal et al. 2011), immunofluorescence (Cheville 1966b), and PCR (Jindal et al. 2015;Medaglia et al. 2015;Riyesh et al. 2016).
Although the causative agent of swinepox was identified a century ago, the molecular epidemiology of the disease is poorly understood. Very recently only, three whole genome data have been published i.e. in addition to the existing USA SWPV reference sequence , one from India (Aasdev et al. 2021) and two from Germany (one each from domestic and wild pig) (Kaiser et al. 2021). SWPV is comprised of a central coding region (139 kb) and inverted terminal repeats (3.7 kb each). Further, the genome can be demarcated as core or central conserved regions (ORFs 21 to 125) and variable terminal regions (ORFs 1 to 20; and 126 to 150). The genes in the core are mainly involved in the replication process and genes in terminal regions are important in determining host range, immune evasion, virulence, etc. Delhon et al. 2007;Bratke et al. 2013). The name host range genes are mainly related to the tropism of cell culture rather than the host system; the host range genes are dispensable for the permissiveness of cell culture but require growth in animals (McFadden 2005;Haller et al. 2014;Oliveira et al. 2017). About 12 groups of host range genes are identified from different poxviruses but none of them is common for all the genera (Oliveira et al. 2017).
The host range genes of SWPV largely overlap with those found in other clade II poxviruses, but they have a unique gene profile, with the notable absence of orthologs of T4, ANK/F-box group 6, and TNFR-2-related genes found in other clade II poxviruses (Bratke et al. 2013). In comparison with the related LSDV, 13 genes (including the T4 ortholog) are missing in SWPV . Work on SWPV host range genes has been very limited and therefore, the present study was aimed to characterize the Indian SWPV at genetic and phylogenetic levels from the two swinepox outbreaks which occurred one each from Haryana and Uttar Pradesh states of India, respectively. The scabs samples from the animal showing pox lesions were collected and genetically and phylogenetically characterized by targeting five host range genes (ORFs 01, 05, 07, 119, and 120) for the Haryana outbreak and two host range genes for the UP outbreak (ORFs 01, and 119).

Sample collection and processing
In October 2015, there was a suspected swinepox outbreak in commercial pig farms near the town of Nilokheri, Karnal District, in the Indian state of Haryana. A team from the Division of Virology, ICAR-Indian Veterinary Research Institute (IVRI), Mukteswar, Uttarakhand, India visited the affected farms on 6 October 2015. The affected animals in this outbreak belong to Yorkshire White pigs. The team also investigated another outbreak that occurred in April 2016 on a pig farm located in Bareilly District of Uttar Pradesh. In the latter outbreaks, the animals affected were Landrace cross, and Desi (local non-descriptive black) breeds ( Table  1). The scab samples were collected and transported to the IVRI in Mukteswar for virological and molecular examinations. The outbreak location map is depicted in Figure 1.

Method
The primers used for the PCR and sequencing of SWPV genes are listed in Table 2. The primers were designed using primer 3 PLUS software with reference sequences available in GenBank (NC_003389.1). Clinical samples were homogenized in phosphate-buffered saline (PBS, pH 7.2) to generate a 10% suspension and then treated with a double dose of Antibiotic Antimycotic Solution (Himedia, India). DNA from clinical samples was extracted using a modified FNES (fast, non-enzymatic, and simple) protocol described previously (Santhamani et al. 2013). The quantity and purity of the DNA obtained by this method were determined using a NanoDrop spectrophotometer.
For the genetic characterization studies, five genes (ORFs 01/150, 05/149, 07, 119, and 120) were targeted for the Haryana outbreak and two host range genes (ORFs 001, and 119) for the UP outbreak. The PCR was carried out using Terra TM PCR Direct Red Dye Premix (TAKARA, Mountain View, CA, USA) in a total volume of 25 lL, and the thermal conditions were as follows: initial denaturation at 98 C for 2 min followed by 35 cycles of 98 C for 10 s, 30 s and 68.0 C for 1 min, with a final extension at 68.0 C for 5 min; the annealing temperatures for the different PCRs are provided in Table 2. To obtain the sequence for a full length in addition to the sequencing of PCR amplicons directly (gel purified), the PCR products were also sequenced by cloning protocol using the pTZ57R/T vector. After confirmation by the gene-specific PCRs, the cultures containing the positive clones (at least two clones per sample) were prepared as stab cultures and sequenced commercially (SciGenom, Cochin, Kerala, India).

Data analysis
Representative nucleotides and deduced amino acid sequences of SWPV were downloaded from the NCBI database and used for comparative analysis. Swinepox Reference Sequence NC003389.1 (GenBank AF410153.1) was used to indicate the positions of nucleotide and amino acid. The multiple alignment and phylogenetic analyses were carried out in MEGA XI software. The phylogenetic tree was constructed using the Neighbor-Joining method with 1000 replicates (Bootstrap method). The evolutionary distances were computed using the K2P method (number of base substitutions per site). The rate variation among sites was modelled with a gamma distribution (shape parameter ¼ 1). All ambiguous positions were removed for each sequence pair i.e. pairwise deletion option (Tamura et al. 2021).

Results
In the present study, investigations were carried out from two farms one each from UP, and Haryana States. In the UP outbreak, both Landrace crossbred and Desi black (nondescript) breeds. Extensive pox lesions spread over the entire body in Landrace cross and both male and female animals were infected.  Though adult animals are considered resistant to infection, severe lesions could be observed in both young and adult animals. Due to the hair and colour of the Desi breeds (black), the visibility of the lesions was low; however, lesions could be observed in the snout, leg and abdominal area (Figure 2). In the Haryana outbreak (Yorkshire white pigs), the severity was comparatively low and lesions appeared larger but with few papules; both male and female young animals showed the lesions but not in adult animals ( Figure 3). The samples collected from Haryana were screened with five host range genes (ORFs 01, 05, 07, 119, and 120) and two host range genes (ORFs 001, and 119) for samples from UP. The category of host range gene/functions is provided in Table 2. The comparative nucleotide and deduced amino acids analyses showed several single nucleotide polymorphisms (SNPs), and a few indels (deletion, and insertion) between the European-North American lineage and Indian lineage. The results of all five ORFs are presented in Table 3; graphical visualization is depicted in Figures S1 to S10 (Supplementary material) and the results are briefly discussed below. ORF001 (ORF 150 is an identical copy) encodes an A52R family protein (Delhon et al. 2012) that likely functions in modulation or evasion of host immune responses, modulation or inhibition of host cell apoptosis, or cell or tissue tropism (Massung et al. 1993;Tulman et al. 2002). The length of the ORF001 for European-North American lineage is 453 nts with a coding capacity of 150 amino acids, while the Indian SWPV sequences had an extension of nine additional nts due to the frameshift in the expected stop codon which resulted in a length of 462 nts with 153 deduced amino acids. Differentiation markers could be observed between these two lineages at both nucleotide and amino acids levels. A total of 18 nts (17 single nucleotide polymorphisms -SNPs and a stretch of 9 nts insertional indel) lead to 11 amino acid differentiation markers (Table 3; Figures S1 and S2). The ORF001 sequence also showed significant homology with other capripoxviruses (data not shown) and caution is required when developing diagnostic assays based on the ORF001 gene or recombinant protein.
Another host range gene targeted was ORF005 (C3L gene; ORF 146 is an identical copy) which encodes a 269 amino acid polypeptide (G proteincoupled CC-chemokine-like protein GPCR) with a putative molecular weight of 31.5 kDa. The length of ORF005 of the European-North American lineage was 810 nts, while the Indian lineage had 813nts. This difference in size was due to the insertional indel of three nucleotides (AAT) between the nts 309 and 310. Therefore, the Indian lineage is expected to code for 270 amino acids. Overall, 20 differentiation markers (19 SNPs, and 1 insertional indel) at nt level and 10 markers at aa level have been observed   A36T, C56T, T88G, C100T, A101G, G114A,  T189C, G226T, T228G, A272G, T291C, T297C,  T319G, C367T, G424A, T429C, G448T, A453T (this is a stop codon for western isolates whereas frameshift in Indian isolates leads to the extension of 9 additional nts (AGGGATTAA). Therefore, the length of this gene is 453 for western and 462 for Indian SWPV sequences.
11 markers L12F, T19M, L30V, H34C, A76S, K88R, Y91C, S107A, A142T, G150C. A total of 150 aa in European-American isolates whereas 3 additional aa at the terminal (YRD) in Indian SWPV due to frameshift in the expected stop codon.  between these two lineages (Table 3 and Figures S3  and S4). The third targeted host range gene was ORF007 which encodes the A52R family protein (Alcam ı and Smith, 1992). Both the lineages had an ORF length of 711 bp with a coding capacity of 236 amino acids. Overall, differentiation markers were observed in 17, and 5 locations at nt and aa levels, respectively (Table 3; Figures S5 and S6).
ORF119 encodes a polypeptide of 185 amino acids (extracellular enveloped virus proteins -EEV glycoprotein). The expected length of the gene is 558 nts with a coding capacity of 185 amino acids and both the lineages showed the same length and coding capacity. There were, 12 nts and 6 aa differentiation markers observed between the two lineages (Table 3; Figures S7 and S8).
ORF120 encodes a polypeptide of 169 amino acids (extracellular enveloped virus protein) which is associated with virulence (Massung et al. 1993). The length of the gene of the European-North American lineage is 510 nts which encodes 169 amino acids. However, the Indian lineage had 513 nts, with a coding capacity of 170 amino acids. This size difference is due to the insertional indel of three nucleotides (AAT) between positions 396 and 397 (N). Overall, there were 14 nts and 4 aa differentiation markers observed between the two lineages (Table 3; Figures  S9 and S10).
The BLAST analyses of the above five genes reveal that all the Indian SWPV (Indian lineage) shared more than 99% nts identity within them. Similarly European and North American SWPV (European-North American lineage) shared >99% identity.  However, there was a significant divergence between SWPV of India and European-North American lineage. It was observed that 95.39%, 96.92%, 97.47%, 97.85%, and 97.08% nts identity for ORFs 1, 5, 7, 119, and 120, respectively which correlated with the SNPs and indels observed between the Indian and western SWPV sequences.
The phylogenetic tree with all the five genes used in the current study deciphered that there are two distinct clades of SWPV viz., the Indian SWPV lineage and the European-North American SWPV lineage (Figures 4-8). The latter clade includes sequences from USA, German, and Russia (available for a few genes only).

Discussion
The present investigation reveals that all breeds of pigs are susceptible though the severity, size and type of cutaneous lesions may vary. In the previous studies also multiple cutaneous lesions were observed from different parts of the body (McNutt et al. 1929;Kim and Luong 1975;Olufemi et al. 1981;Jubb et al. 1992). In congenital infections, lesions may appear on the entire body and in the case of mechanical transmission by the vector, the lesions can be found in the feeding areas of the vector (Delhon et al. 2007). Further, it was reported that both domestic and wild pigs can be infected by SWPV. House flies are one of the possible mechanical vectors, and the presence of these flies is presented on both farms. Therefore, is difficult to conclude the source of infections. In a recent swinepox outbreak in Germany, the farms practiced all the hygienic measures and therefore mechanical vector transmission is unlikely; the transmission might have occurred through persistently infected asymptomatic animals (Kaiser et al. 2021).
After the first complete SWPV genome was published in 2002 ), a few months ago only, three additional sequences were published viz., one from India (domestic pig) (Aasdev et al. 2021) and two from Germany (one each from domestic and wild pigs) (Kaiser et al. 2021). In addition, a few host range genes of SWPV are also characterized by Indian researchers (Riyesh et al. 2016). The availability of sequence data enhanced our understanding of the biology and epidemiology of SWPV from different countries.
The DNA was extracted using the modified FNES protocol (Santhamani et al. 2013). The concentration obtained was 25 ng/lL per sample and the 260/ 280 ratio was above 1.8, indicating that the DNA had a high degree of purity. This protocol has been already employed for other pox viruses like goatpox and sheeppox (Ramakrishnan et al. 2017) now we have incorporated it successfully for the swinepox virus also.
Recent three studies enhanced our knowledge of the evolutionary relationship of SWPV from different countries. In one study, analyses of host range genes of SWPV (Riyesh et al. 2016) reveals that Indian SWPVs are genetically closely related to SWPV of the USA. In 2021, the first whole genome sequence of Indian SWPV was carried out and it was opined that the Indian isolate belongs to Eurasian lineage and diverges significantly from American lineage (Aasdev et al. 2021). Almost in the same period, interesting data were obtained from the whole genome sequencing of SWPV from domestic and wild pigs by researchers from Germany (Kaiser et al. 2021). Their study indicates that the USA and German isolates clustered under one lineage whereas Indian SWPV formed a separate lineage; further differences could be observed between some of the ORFs of domestic and wild pig origin (Boh orquez et al. 2021). It was hypothesized that the two lineages of SWPV could have evolved at a certain point of time during the domestication of pigs (Aasdev et al. 2021;Kaiser et al. 2021). Our study affirms that Indian SWPVs are distinct from European-North American lineage. Need more data to understand either lineage-level classification is sufficient or need to classify them into species levels. In the Genus Capripoxvirus, there are three species viz., sheeppox, goatpox, and LSDV of cattle were recognized based on the sequencespecific markers in some genes and/or the entire genome (Ramakrishnan et al. 2017). The comparative pathogenicity, immunogenicity, and vaccine efficacy of both lineages will shed a plethora of information on SWPV.
In conclusion, all five host range genes of the Indian SWPV sequences showed significant divergence from the SWPVs of European-North American SWPV sequences due to the high variations at both the nucleotide and amino acid levels. Although the two outbreaks occurred several miles apart, the molecular epidemiological studies indicate that the Indian SWPV might have originated from a single gene pool. The findings of the present study will help in better understanding of the molecular epidemiology of the SWPV circulating in India and aid in the development of vaccines and nucleo-diagnostic tools.