Phylogenetic analysis of Jaagsiekte sheep retrovirus (JSRV) in Iraqi Awassi sheep

Jaagsiekte sheep retrovirus (JSRV) infection in sheep results in contagious ovine pulmonary adenocarcinoma (OPA). The aim of this study was to detect the presence of JSRV in Iraqi Awassi sheep of Al-Qadisiyah Province using reverse transcription-PCR (RT-PCR). Lung secretion samples from 50 adult sheep (1.5 to 2-year-old) during 2016-2018 that were suffering from chronic respiratory distress with suspected clinical OPA were collected. RT-PCR assay for detection of JSRV was conducted using primers specific for its 382 bp envelope protein gene. The results showed that 22 of the 50 (44%) lung secretion samples were positive for JSRV. Phylogenetic tree analysis revealed the JSRV sequence detected in the local Iraqi sheep population was closely related to NCBI BLAST locus isolates KT279066.1 and KT279065.1. The study found that the JSRV disease is prevalent in the Iraqi Awassi sheep flocks and that the local JSRV isolate is genetically divergent from most other global isolates.


Introduction
Jaagsiekte sheep retrovirus is a Betaretrovirus and its genomic organization comprises only the important genes typical of retroviruses: gag, pro, pol, and env (1). It is thought that after the first viral infection, the virus may persist in white blood cells for few years before inducing neoplastic lesions by infecting pneumocytes and club cells upon reaching the lungs (2). During the 2017 epidemic OPA in Awassi sheep of Iraq, the first molecular and histopathological study occurred (3). Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is characterized by the unvaryingly serious lung tumors that appear principally in mature sheep. OPA has a world-wide prevalence (4); as reported by the International Epizootic Office, it may have spread worldwide through international trade (5).
Greater incidence and infection rates are observed during the initial infection of the farm sheep, causing high economic losses in the farms. JSRV is an oncogenic retrovirus that affects epithelial cells of the lungs, especially the type II pneumocytes and club cells, and the initial infection proceeds to the formation of invasive bronchioloalveolar carcinoma (6). The clinical signs of JSRV infection are progressive emaciation and exerciseinduced chronic respiratory distress; the affected animals often lag in the flock, and thin mucus secretions from nostrils is observed. When the head is lowered, a copious foamy exudate or discharge from both nares occurs, and a moist rale is heard. However, coughing is not typically noticeable during auscultation (7). The clinical symptoms progress gradually, culminating in severe dyspnea; frequently, pneumonia due to secondary bacterial infection can occur, followed by death in a few days (8,9).
In the presence of clinical respiratory signs or tumors, the diagnosis of OPA (10) and the detection of JSRV in lung secretions by RT-PCR (11) is easy. However, throughout the pre-clinical stage, it is difficult to detect the infection because of the absence of detectable circulating JSRV proteins and JSRV-specific antibodies external to the tumor (12). Thus, pre-clinical findings of JSRV infection in routine examinations are not possible.
The aim of this a study was to first report on the sequencing and phylogenetic analysis of JSRV in Iraqi Awassi sheep.

Ethical approval
The present study has approved by the Committee of Ethics of the College of Veterinary Medicine, University of Al-Qadisiyah, Iraq.

Specimen collection
A total of 150 animals from Awassi sheep were clinically inspected for the presence of clinical signs of JSRV infection, Lung secretion samples were collected randomly from the 50 adult affected animals 1.5-2 year old during 2016-2018 that suffered previously from emaciation, chronic respiratory distress and non-responsive to treatment with antibiotics, and were suspected to have JSRV infection. Collected samples were immediately stored under sterile and cold conditions 2-8ºC until the RT-PCR assays and additional tests.

RNA extraction
Viral RNA extractions from lung secretions were performed using the RNA extraction AccuZol TM" kit (Bioneer, Korea). In brief, 200 µl of the lung secretion sample and 1 ml of Trizol reagent were mixed together on a vortex for 1 min, in a 1.5 ml Eppendorf tube. Subsequently, 200 µl of chloroform was added and mixed well for 15 Sec. Following 5 min of incubation on ice, the mixture was centrifuged at 12000 rpm for 15 min at 4ºC. The supernatant was then transferred to a new tube and 500 µl of isopropanol was added. The mixture was mixed by turning the tube over for 4-5 times and was incubated for 10 min at 4ºC.
The mixture was then centrifuged for 10 min at 12000 rpm, and the supernatant was discarded. Subsequently, the RNA pellet was washed with DEPC, resuspended in 1 ml of 80% ethanol, and re-pelleted by centrifuging at 12000 rpm for 5 min at 4ºC. Next, the supernatant was discarded and the RNA pellet was air-dried. Finally, 50 µl of DEPC water was added for the elution of the RNA pellet, and tested by using NanoDrop (Bioneer, Korea). The extracted RNA was stored in a -20ºC freezer until RT-PCR was carried out.

Reverse transcription-PCR
Reverse transcription-PCR (RT-PCR) was carried out using a one-step RT-PCR kit (Bioneer, Korea, AccuPower ®) RT-PCR PreMix). The specific primers, used for RT-PCR amplification of the gene encoding JSRV envelope protein was designed from the retrovirus isolate GVM-7 (envelope protein gene), partial CDS GenBank: (KT279065.1). The specific primer, and reverse transcription-PCR master mix was prepared according to the manufacturer's instruction summarized (13).
PCR amplification and reverse-transcription were performed in a one-tube reaction under the following thermo-cycler conditions: reverse transcriptase, one cycle at 50ºC for 15 min; initial denaturation, one cycle at 50ºC for 15 min; denaturation, 40 cycles at 95ºC for 20 sec; annealing, 40 cycles at 58ºC for 30 sec, final extension, 72ºC for 5 min; and a final hold at 4ºC. PCR product bands were visualized using a UV transilluminator (Vilber Lourmat, France).

Sequencing and analysis
PCR end products was sent to Bioneer, Korea for sequencing of the cDNA of the retrovirus using a specific (AB-DNA) sequencing system (gpB) forward primer. The resulting sequence and those of other global isolates obtained by NCBI blast search were aligned by the Arithmetic mean method and MEGA 7.0 to construct a phylogenetic tree.

Results
In this study, RT-PCR was used for the detection of (JSRV) using specific primers to produce 382 bp JSRV envelope protein gene. It was found that 22 out of the 50 lung secretion samples tested were positive for JSRV ( Figure 1).
The nucleotide sequences of the local isolate were crosschecked using NCBI BLAST, to validate the RT-PCR result. The phylogenetic tree analysis revealed that the local Iraqi JSRV was closely related to NCBI BLAST locus Indian isolates KT279066.1 and KT279065.1, while other JSRVs were located out of the tree (Figure 2).  Based on the NCBI BLAST sequence query alignment, the local Iraqi isolate of (JSRV) showed a high identity (100%) with an Indian strain (KT279065.1 and KT279066.1), and 98% sequence similarity with UK strain (MF175071.1) however divergent from other isolates.

Discussion
OPA was first recorded in Africa in 1825 (3), and was subsequently found among flocks in various countries (14,15). The first molecular and histopathological recording of OPA in Iraq occurred during the 2017 respiratory distress epidemic cases and confirmed the occurrence of JSRV in different areas of Al-Qadisiyah Province. Moreover, the molecular results confirmed JSRV occurrence in Iraqi Awassi sheep (15,16).
These recent studies were in agreement with (17)(18)(19). In the current study, samples were collected from affected sheep based on the typical clinical features of OPA. Using RT-PCR technique, the 382-bp JSRV DNA was identified in lung secretion samples obtained from adult sheep that were clinically identified to be suffering from the respiratory disorder. This result is in agreement with other studies (20,21) that used PCR assay to diagnosis this virus. Eradicating OPA from a flock is difficult, because there is no available cost-effective assay for the detection of this virus and the infection cannot be detected in the preclinical stage by most diagnostic tests (22,23).
The NCBI BLAST-based sequence enquiry showed that the Iraqi isolate evaluated in this study was closely related to the Indian isolates KT279066.1 and KT279065.1. We think that this may be due to the importation of sheep from India and other countries to Iraq.
Analysis of the phylogenetic tree revealed that, although the local JSRV isolate has high sequence similarity, is closely related, and clusters together with global isolates such as MF17507.1, EF680317.1, and KT279067, the local JSRV isolate exhibits divergence with other isolates (XM-01502802.1 and XM-015102072.1), which were located outside the generated phylogenetic tree. This finding is in agreement with previous reports (24,25) .

Conclusions
In conclusion, this study was the first report on sequencing and phylogenetic analysis of JSRV strains in Iraqi Awassi sheep which, will be of use for prospective studies of JSRV in Iraq, besides using lung secretions could be useful in the detection of disease by PCR.