Accession numbers of study isolates
The partial length GIF/IL-2 gene (408 bp) was successfully isolated in 58/441 animals (51 goats, 7 sheep) (Fig. 2). The detail of isolated samples from different districts is listed in Table 1. The nucleotide sequences of the current study were submitted to NCBI GenBank Database and are available under the following accession numbers; MZ054288 (orfV/Lodhran/1/Goat/2020/Pakistan), MZ054291 (orfV/Lodhran/5/Goat/2020/Pakistan), MZ054297(orfV/Muzaffargarh/18/Sheep/2020/Pakistan), MZ054293 (orfV/Dera Ghazi Khan/8/Goat/2020/Pakistan), MZ054285 (orfV/Multan/20/Goat/2020/Pakistan), MZ054292 (orfV/Dera Ghazi Khan/6/Goat/2020/Pakistan), MZ054299 (orfV/Lahore/22/Goat/2020/Pakistan), MZ054286 (orfV/Lahore/23/Goat/2020/Pakistan), MZ054281 (orfV/Dera Ghazi Khan/9/Goat/2020/Pakistan).
GIF/IL-2 gene sequence analysis
The percentage identity matrix of GIF/IL2 gene of study isolates and other parapoxvirus retrieved from NCBI GenBank were demonstrated using similarity (%) and pairwise distance matrix and tabulated separately for nucleotide (nt) (Table 2) and amino acid (aa) (Table 3). The results revealed that the Pakistan isolates had 94.4%~99.9% and 96.7%~99.9% similarities at nt and aa level, respectively. Moreover, nucleotide homology (%) of the study isolates with the reference strain of ORFV (NC_005336) is represented in Fig. 3a, which shared maximum nucleotide (97.2~98.7%) and amino acid (97.6~100%) sequence homology. Similarly, ORFV originated from neighboring countries (India and China) exhibited the highest identities, 98.1%~99.9% and 99.2%~99.9% at nt and aa levels, respectively (Fig. 3b). The nucleotide and amino acid sequence analysis revealed a close relationship between the study and neighboring countries isolates.
Amino acid substitutions
The alignment of amino acid sequences of GIF/IL2 gene of the study isolates with NCBI GenBank reference strain, ORFV/OV-SA00/Goat/USA/2003 (NC_005336), is shown in Fig. 4a. Six isolates (MZ054297, MZ054293, MZ054285, MZ054292, MZ054299, and MZ054286) have no unique amino acid substitutions; however, the isolates (MZ054288, MZ054291) have only one substitution (R5W), while isolate (MZ054281) has three unique amino acid substitutions (S16N, H18P, D103N) with the reference strain. Analysis based on ORFV-GIF/IL2 gene of neighboring countries (India, China) revealed that Indian ORFV-isolates (DQ922634, KY077477, KY077478) have no amino acid substitutions with the study isolates. In contrast, Chinese ORFV-isolates (MF489140, MF489145, MF172958, KF726847, KF666566, MF489142) have unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with the study isolates (Fig. 4b).
Phylogenomic and evolutionary tree
The phylogenetic analysis based on GIF/IL2 gene of 51 PPVs sequences revealed that ORFV lineages were broadly grouped into five distinct clusters/clade with >75% bootstrap support (Fig. 5). Cluster I comprises goat-originated Asian ORFV isolates, while clusters II, III, and V include Chinese, Indian, and Iraq-originated sheep ORFV isolates. Muskox and mountain goats (USA) derived ORFV isolates were placed in a monophyletic clade (cluster IV). The isolates from Pakistan clustered together with the Chinese (AH1404, Xinjiang1, AH1612, AH-GY13, Shihezi3, Xinjiang2, AH1508, GZ18) and Indian isolates (MUK59/05, MLMR/TN, MEC/TN) into the same clade (cluster I). Also, an evolutionary tree based on GIF/IL2 gene sequences segregated the PPVs into three distinct groups; ORFV, PCPV and BPSV, representing a close relation between ORFV and PCPV in evolution, compared to BPSV (Fig. 6).
Pre-requisites for Case Definition for ORFV in Pakistan
Clinical signs and symptoms
Diseased animals exhibited different clinical manifestations such as anorexia, starvation, emaciation, decreased weight gain, depression, and dullness. The lambs and kids were severely affected due to restricted suckling and grazing. Female animals also have developed teat crusts, which may be associated with oral cavity lesions of suckling. Furthermore, some animals developed respiratory problems, which could be due to secondary bacterial infection or aspiration pneumonia caused by bottle-feeding. Moreover, ORFV promoted death in young animals and myiasis in adults, primarily observed in a warm climate.
Gross lesions
The infected animals had the typical multifocal to coalescing, ulcerated lesions on the epidermis of gums, lips, mouth commeasure, muzzles, nose, and udder (Fig. 7a, 7b, 7c). Also, infected animals had severe proliferative papules, pustules, and crust formation (Fig. 7d, 7e). However, some animals also had the advanced stage in thicker, brownish, quickly accretive scabs with granulation (Fig. 7f, 7g, 7h). In addition, some animals exhibited the healing and regeneration stage of lesions (Fig. 7i).
Histopathological and Microscopic analysis
Histopathological examinations revealed epidermal hyperplasia with hyperkeratosis and parakeratosis with prominent thickening of epidermis resulted in acanthosis (Fig. 8a). Epidermal degeneration is characterized by lysis of epithelium replaced by a multifocal-to-coalescing necrotizing sero-cellular crust (Fig. 8b). The epidermis extends into the dermis and forms anastomosing rete ridges (Fig. 8c). Spinous cells in the stratum spinosum showed degenerative changes, including spongiosis and vacuolation with pyknotic and karyorrhexis nuclei (Fig. 8d). The epidermis contained peripheralized keratohyalin granules (Fig. 8h), micro abscess, and scab formation (Fig. 8e). Typical intraepithelial ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies were detected in keratinocytes (Fig. 8g). The superficial dermis is expanded by infiltration of inflammatory cells (Fig 8f), intracellular edema and scattered hemorrhages (Fig. 8i).