A Comparative Study on Global Genetic Diversity and Population Genetic Analysis of Orf Virus Isolates from Outbreaks and it’s Implications for the Vaccine Development

Contagious ecthyma (Orf, contagious pustular dermatitis, infectious labial dermatitis, scabby mouth, sore mouth) is a viral disease that is caused by the epitheliotropic Orf virus and it primarily causes disease in goats, sheep, and other ruminants globally [1-3]. The disease is also transmissible to humans (zoonotic) due to direct or indirect contact to people working with animals [4] and infections are manifested by painful lesions in hand and can spread to other organs [5]. The disease in sheep and goats are characterized by inflammatory, proliferative and scabby lesions in the lips, oral and nasal mucosa and muzzle. The lesions are painful and depending on the location of the lesions, animals may be unwilling to nurse, eat or walk [6]. Although, vaccination Abstract

Copy@ Saeed Nazeri with attenuated Orf virus is the most common method to prevent and control the disease [7,8] there are recent reports revealing that the vaccination with the live attenuated virus cannot provide effective protection [9] suggesting that the Orf virus strains might have been changed genetically [10][11][12]. The virus is a member of Parapox virus genus which belongs to the Poxviridae family. It has a linear double-stranded DNA genome (134-139kb in size) [13]. The central region of the genome encodes proteins involved in the virus structure and assembly, while in the terminal regions of the genome there are genes associated with virulence, pathogenesis, host range [14,15]. The envelope gene (B2L) of the virus encodes a highly immunogenic major envelope protein of molecular weight about 42kDa and exhibits lipase activity [16]. This gene has been widely used for molecular characterization and phylogenetic analysis of isolates of Orf virus [8,17,18].
In recent years, outbreaks of Orf virus have occurred worldwide [12]. Although outbreaks of Orf virus have occurred in Iran and have been confirmed, there is no report available of the detailed sequence and phylogenetic analysis of the B2L gene of Orf virus in Iran in comparison with other isolates from other regions of the world.
Having this data may resolve the determinants of gene flow and patterns of genetic diversity in global endemic regions. Therefore, in the current investigation, the main objective was to investigate the sequence diversity and population genetic of the B2L gene among natural Orf virus isolates. On the other hand, we extracted worldwide B2L sequence data available in the GenBank, and along with Iranian B2L sequence data, the global genetic diversity of this gene was evaluated. The obtained results would add more data on available global information regarding B2L genetic diversity and would also allow comparing the Orf virus population from this part of the world with those found in distinct and contrasting epidemiological settings to identify polymorphisms. The results of this study may be helpful for developing an appropriate live attenuated contagious ecthyma vaccine.

Sampling of Orf Virus from Sheep and Goats
In the current investigation, 24 mouth scab samples were  Table 1). The outbreaks were characterized by the presence of cutaneous lesions on lips with no mortality. Sampling was performed using sterile forceps individually for each animal and the samples were immediately immersed into sterile PBS supplemented with 1% antibiotics (Penicillin 1000 IU-Streptomycin 10mg; Sigma, Germany). The samples were chilled on ice during the transport to the laboratory of Vira Vaccine Co. and were stored at -20°C for subsequent use.  IRTN1  2018  Taleqan  Sheep  3  MN422309   IRTN2  2018  Taleqan  Sheep  4  MN422310   IRTN3  2018  Taleqan  Sheep  3  MN422311   IRTN4  2018  Taleqan  Goat  1  MN422312   IRTN5  2018  Taleqan  Goat  2  MN422313   IRTN6  2018  Taleqan  Goat  2  MN422314   IRTN7  2018  Taleqan  Goat  1

Virus Isolation
The collected mouth scab samples were washed three times with sterile PBS, cut into small pieces, ground, and suspended (1:10) in sterile PBS supplemented with antibiotics. The suspension was centrifuged at 5000 rpm for 10min and the supernatant was used to infect primary lamb testis (LT) cells.

Amplification and Sequencing of B2L Gene
For molecular analysis of B2L gene, PCR amplification was performed using a forward outer primer (B2LF: 5′-GACCTTCCGCGCTTTAATTT-3′) and a reverse primer (B2LR: 5′-CCCGCCTGCTAAAAGACT-3′) [19].  were also used to identify the departure from neutral patterns of nucleotide substitutions as deviations between the estimates of θ derived from the number of phylogenies compared to either the total number of mutations (D*) or the average pairwise diversity (F*) [24]. A positive value of Tajima's et al. [24,25] D tests corresponds to positive natural selection, whereas a negative value signifies an excess of low-frequency polymorphisms relative to expectation, indicating population size expansion and/or negative/purifying selection. To examine whether deviation from neutrality occurs at certain residues of B2L, a sliding window analysis of Tajima's D [25] across the coding sequences was done using a window size of 250bp and a step size of 10bp [26].

Population Genetic Analysis
The genetic difference among the isolates of different endemic regions was measured by the fixation index (Fst) and calculated from the average number of pairwise DNA sequence diversity interspecific and intraspecific populations using DnaSP software.

Identification of Orf Virus
To confirm the Orf virus identity, total DNA was extracted from the mouth scab samples and screened by PCR amplification. days in culture for either the first or second blind passage ( Figure   3A). In the negative control no CPE was observed ( Figure 3B).

Sequence analysis of Iranian and global B2L gene sequences
In the current study, the complete B2L gene (24 samples) were amplified, sequenced and used for comparative analysis.    show evidences of departure from neutral expectation (Table 4).

Genetic Differentiation Between Global Orf Virus and Phylogenetic Tree Construction
We observed population differentiation between isolates from Alborz, Tehran and Kerman provinces in which the FST values

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Conclusion
The present analysis has shown for the first time, a genetic