Isolation and characterization of lumpy skin disease virus from cattle in India

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells, an alternative cell line that was found suitable for LSDV propagation. In addition, kinetics of viral DNA synthesis and one-step growth curve analysis suggested that LSDV initiates synthesizing its genome at ∼24 hours post-infection (hpi) with a peak level at ∼96 hpi whereas evidence of progeny virus particles was observed at 36-48 hours (h) with a peak titre at ∼120 h. This study reports the first successful isolation of LSDV in India, besides describing alternative cells for virus isolation (primary goat kidney cells) and in vitro propagation (Vero cells) as well as providing some insights on LSDV life cycle. Author Summary This study describes the first successful isolation of LSDV in India. Nucleotide sequencing of LSDV ORF011, ORF012 and ORF036 suggested that the isolated virus is closely related to Kenyan LSDV strains. This is in accordance with the previous (only) study, thereby suggesting that a single LSDV strain is circulating in India. For the first time, we employed primary goat kidney (PGK) cells for the isolation of LSDV, where PGK cells were found more sensitive for LSDV isolation as compared to the primary lamb testicle (PLT) and primary goat testicle (PGT) cells. We also adapted the isolated virus in Vero cells and demonstrated that Vero cells are also suitable for in vitro propagation of LSDV. Besides, we also provide some insights on the LSDV life cycle.

2 4 73 thickness hole in the skin which usually gets infected by bacteria or becomes liable 74 to myasis [8]. Some animals become extremely emaciated, and euthanasia may be 75 warranted. Besides, the bulls may become temporarily or permanently infertile and 76 may secrete the virus for a prolonged period. The morbidity in LSD varies from 50-77 100% [9]. The mortality rate is usually low (1-5%) but occasionally reported to be 78 much higher [10]. 79 The occurrence of LSD causes decreased milk production, loss of hide and Poxviridae. LSDV genome is ~151 kbp in length [12]. Two other capripoxviruses, 88 sheepox virus (SPV) and goatpox virus (GPV) which cause devastating disease in 89 sheep and goats respectively, are also antigenically similar to LSDV [13]. 90 Capripoxviruses are cross-reactive within the genus; therefore SPV-or GPV-based 91 vaccines have been used to provide cross protection against LSDV [13][14][15]. LSDV is 92 usually isolated and quantified (tissue culture infective dose; TCID 50 ) in primary cells. 93 LSDV also infects Madin-Darby bovine kidney (MDBK) cells where it forms foci 94 (multifocal areas of hyperplastic cells). These foci can be counted under microscope 95 in an agar-overlay medium. However, a plaque assay to precisely quantify infectious 96 LSDV is still lacking.

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The recent and unprecedented spread of LSD in India and several other 98 countries has highlighted the need for better research efforts into this rapidly 99 emerging pathogen. Our study describes alternative cells for isolation and in vitro 100 propagation of LSDV, besides providing insights on LSDV life cycle.

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Ethics Statement 103 The study involves collection of biological specimens from cattle (field animals). 104 Scabs and blood samples (3 ml each) were collected from the LSD suspected cattle 105 (n=22) as per the standard practices without using anaesthesia. College of 106 Veterinary Sciences, Birsa Agricultural University, Ranchi (India) granted the 107 permission to collect the biological specimens. A due consent was also taken from 108 the farmer (animal owner) before collection of the specimens.  Tussum, Nagri, Khemra and Gadgaon. The first evidence of the disease was 119 reported about 2 months prior to the sampling. Scabs from the nodular lesions were 120 collected in 3 ml Minimum Essential Medium (MEM, transport medium) and 121 transported on ice to the laboratory. The samples (scabs) were triturated to make 6 122~10% suspension in MEM followed by filtration through 0.45 µM syringe filter and 123 storage at -80 O C until use. Serum/blood samples were also collected from infected 124 as well as apparently healthy animals after taking a due consent from the farmers.

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Identification of the agent (s) 126 Typical nodular lesions on the body surface were primarily suggestive of LSD.

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Therefore, initially we investigated for the presence of capripoxvirus and LSDV-128 specific gene segments by PCR (Table 1)   painful and were 1-3 cm in size (Fig. 1b). Some of the nodules ruptured to create a 208 deep-seated wound (Fig. 1c, 1d). Wounds were frequently invaded by secondary 209 bacterial infections which led to extensive suppuration and sloughing. The lesions 210 were particularly extensive in the fetlock region, extended up to the underlying 211 subcuitis and muscle (Fig. 1e). Some of the nodules regressed and in some the 212 necrosis of the skin resulted in hard, raised areas (sit-fasts) clearly separated from 213 the surrounding skin (Fig. 1f). Nasal discharge was apparent only in few animals.  (Table 2). 227 However, viremia could not be detected; neither in the clinically affected animals with 228 skin nodules nor in the healthy in-contact susceptible animals (Table 2). 229 Furthermore, the scabs were positive for LSDV-specific, rather than SPV-or GPV-    consensus linear phylogenetic tree (Fig. 4). Nucleotide sequences of 243 LSDV/India/2019/Ranchi showed highest similarity to the Kenyan LSDV strains.

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Virus isolation 245 The virus recovered from the scabs was used to infect PGT, PGK and PLT cells. was evident at 96-120 hpi (Fig. 5a). The other cell types did not produce any CPE in  (Fig. 5b). Unlike P4, P15 virus also formed plaques but were of small size (Fig. 5c).  Fig. 6a and 6b). 288 In order to provide insights on the kinetics of LSDV genome synthesis, 12 hpi and 18 hpi ( Fig. 6c and 6d). However, the levels of viral DNA progressively 293 increased from 24 hpi to 96 hpi and finally stabilizing at 120 hpi ( Fig. 6c and 6d). 295 Serum samples from all the clinically affected animals (showed presence of 296 skin nodules) and some in-contact susceptible animals were also subjected for 297 determination of anti-LSDV antibody titer in a virus neutralization assay. Clinically 298 affected animals showed an antibody titer of 64-1024 (Table 2). However, few 299 healthy in-contact susceptible animals were also found positive for anti-LSDV 300 antibodies suggesting subclinical infection (