Molecular characterization of circulating strains of the peste-des-petitis-ruminants virus in Sulaimani province, Iraq

Peste des petits ruminants (PPR) are caused by a Morbillivirus that belongs to the family Paramyxoviridae . Peste des petits ruminants are an acute, highly contagious, and deadly disease that mainly affects goats and sheep, with subclinical infection occurring in cattle. Peste des petits ruminants occur in many countries, with morbidity and fatality rates as high as 90 percent. Peste des petits ruminants’ outbreaks occurred annually in Sulaymaniyah; this study aims to determine genetic variation and the global relationship of circulating strains in the area. RT-PCR was primarily used to identify the virus from mouth epithelial samples. The direct sequencing and subsequent analysis of amplified PCR products for the (N and F) genes revealed the presence of PPRV lineage IV in the study areas. Moreover, phylogenetic analysis revealed N gene (OL702851/PPR/H/SUL/sheep, OL702852/PPR/H/SUL2/Goat) cluster with (china-KP319027.1) strain with identity 98%, while the F gene (PPR/H/SUL3/Sheep, PPR/H/SUL4/Goat) has a cluster with (Turkey-MG744218 and Saudi Arabia-MK922470.1) strains with identities 97 and 98% respectably. Furthermore, phylogenetic tree field virus isolates heterologous with the current vaccine strain (Nigeria 75/1) were used for the mass vaccination program, and the divergence rate was 10%-13%. These variations between vaccines and field isolates could compromise the antibody responses induced by the vaccines in the hosts.


Introduction
Peste des petits ruminants (PPR) are a highly contagious viral disease of wild and domestic small ruminants (1), Although cattle, swine, camels, and buffaloes can be infected with the PPR virus (2)(3)(4)(5). Since it was first identified in Ivory Coast (West Africa) in 1942 (6), its geographic distribution has been expanding within Senegal (7), China (8), India (9,10), Pakistan (11), East Africa (12), Egypt (13), Turkey (14,15), Iran (16), Ethiopia (17,18) Bangladesh (19), Nepal (20), Saudi Arabia (21) and Iraq (22). Peste des petits ruminant's virus (PPRV), an RNA virus from the genus Morbillivirus in the Paramyxoviridae family, causes the disease (23)(24)(25). The genome of PPRV consists of 15,948 nucleotides (1) which encode two non-structural proteins, C and V, and six structural proteins arranged in the order nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin (H) and viral RNAdependent RNA polymerase (L) (26). Developing essential molecular diagnostic techniques for PPRV was also aided by sequencing the N and F genes (27). PPRV strains have been classified into four genetically distinct lineages (I, II, III, and IV) based on partial sequences of the N and F genes (28). The acute form of PPR is marked by erosive mouth (erosive stomatitis) with frothy salivation (29), sudden depression, high fever, pneumonia, oculo-nasal discharges, oral necrotic lesions, diarrhea, and dark red spots (congestion) were discovered in several lobes of the lungs, as well as the small and large intestines, during the postmortem examination (30,31). The disease often shows high rates of infertility, and production of underweighted-lambs in young animal, but in mature animal exhibit abortion, stillbirth and lambs born weak, uncharacteristic body shapes and under-sized (32). The virus is predominantly transmitted by oral, nasal, and conjunctiva secretions and stool of the infected animals, which contain high concentrations of virus. The virus is highly infectious and quickly spreads through direct contact between infected animals' secretions and/or excretions and healthy animals nearby (33). Infectious viral infections are hard to control due to a lack of epidemiological data and different management techniques in the herds where the problems occur (34), but the availability of live attenuated PPRV vaccinations that protect animals from recurrent infection is helping to eradicate the PPRV (e.g., Nigeria/75) (35).
The current study's objective was to genetically describe the PPRV isolate in Sulaimani, Iraq, and to compare its sequence to that of the global PPRV and the existing commercial vaccine strain.

Ethical approve
Research proposal was accepted and registered under (No. 11 on 27/ 6/ 2021) by the ethics committee of Veterinary Medicine College/ University of Sulaymaniyah.

Sample collection
Throughout March to July 2021, fifty sick sheep and goats with fever, profuse oculo-nasal discharge, moderate ulcerative stomatitis, dyspnea, coughing, diarrhea, and inappetence had their oral swabs and mouth lesions collected from (Halabja and Penjwen) region in Sulaymaniyah province for RT-PCR detection of PPRV RNA. This study selected (Halabja and Penjwen) districts ( Figure 1). Since the area is mountainous and has a cold environment, especially in the summer, people who own sheep and goats migrate there to raise their animals. These two locations were chosen for sampling for two reasons: first, there is a greater possibility of a disease outbreak because animals from different areas migrate there; second, it borders Iran.

Extraction of RNA
All samples were extracted by using an RNA extraction tissue kit (Genet Bio, Daejeon, South Korea). The extraction had done according to the manufacturer's protocol.

Complementary DNA (cDNA) and PCR
A partial sequence of the (N, and F) gene was amplified by using one-step RT-PCR Premix (2X) (GeNet Bio). The reactions were carried out in a 0.20 mL PCR tube based on the following specifications: 10.00 μL RT-PCR premix, 5.00 μL RNA, 1.00 μL forward (10.00 pmol), 1.00 μL reverse primers (10.00 pmol), and 3.00 μL molecular grade H2O to make up a final volume of 20.00 μL. Partial sequence of F and N genes were amplified separately by using a conventional PCR machine (Hercuvan, Carlsbad, USA) that was programmed as follows: cDNA synthesis 50.00 ˚C for 30 min, initial denaturation at 95.00 ˚C for 10 min followed by 40 cycles of 95.00 ˚C for 45-sec annealing at 58.00 ˚C for 45 sec, and extension at 72.00 ˚C for 1 min and a final extension at 72.00 ˚C for 10 min. Both primers set was the same PCR programmed except in annealing step 60 ˚C for N gen protein. The PCR products were analyzed by loading 7.00 µL on standard 1.00% (w/v) agarose gel (Gendirex, Daejeon, South Korea) in 1X Tris/Borate/EDTA (TBE) buffer. The gel was stained with 4.00 μL safe dye (EURx, Banino, Poland). Electrophoresis was done on 100v for 1 hr. The PCR product was visualized under a UV transilluminator (Uvitec, Cambridge, UK).

Phylogenetic analysis
The PPRV sequences obtained in this study, the sequences of Nigeria/75 vaccine strains available in Iraq, and the sequences retrieved from GenBank were aligned. The evolutionary history was inferred by employing the maximum-likelihood method based on the Tamura Nei parameter model implemented in the MEGA X program (38). The branch lengths were measured regarding the number of substitutions per site. A bootstrap test (1,000 replicates) was done to evaluate the confidence level of branching in the phylogenetic tree.

Result of RT-PCR
Twenty (40%) of fifty samples were positive by agarose gel electrophoretic identification demonstrating an expected amplicon of about 372 bp and 464 bp for F and N genes, respectively ( Figure 2). The results were confirmed by sequencing of PCR product, and the sequences were submitted to NCBI Genebank and released under theses accession numbers (OL702851-OL702854).

Phylogenetic tree and sequences analysis
Identities of 99.30 and 98.80% were observed among the N and F genes of Sulaymaniyah PPRVs, respectively, when analyzing the nucleotide sequences. The analysis of our two isolates for the N gene, which was published in NCBI GenBank, indicated that the 463 bp sequences of two  Table 2). According to phylogenetic tree construction based on the sequence of both N and F gene alignment of PPRV sequences retrieved from NCBI GenBank, all four PPRV isolates belong to lineage IV (Figures 3 and 4). A nucleotide sequence comparison between the vaccines strain (Nigeria/75) currently used for vaccination programs in Iraq and field strains revealed low identities ranging 87-92% for F and N genes, respectively. The phylogenetic tree revealed that PPRV Field strains belong to lineage IV while vaccine strains belong to lineage II. Compared to other sequences, including vaccination strains, multiple sequence alignment field viral sequences for both genes revealed numerous nucleotide substitutions ( Figure 5). As a result, there were few identities and much variance among them.   Figure 3: Neighbor-Joining method phylogenetic tree relationship among PPR isolates. N genes are obtained from sheep, goats sequenced in this study are shown with solid circles (Red), and the vaccine strains are shown with solid rectangles (Green).

Discussion
The field data and laboratory results implicate PPR in the Sulaymaniyah province. Based on the RT-PCR which is the most sensitive assay for confirming PPR diagnosis (39). The ability to distinguish between distinct PPR lineages, more precisely identify the origin of outbreaks, and better comprehend the epidemiology of the PPR virus are all made possible by nucleotide sequencing of the PCR product (40). According to sequencing results, the most likely cause is a PPRV field strain that has been circulating in the region and Sulaymaniyah province for over 10 years. There is a 95-98% similarity between our isolates and the Sulaymaniyah isolates of 2012 (41). Iraq has borders with countries where several economically critical infectious diseases are endemic, particularly Turkey, making it difficult to control the introduction of infectious diseases into the country. As a result, the importance of PPRV as a threat to livestock, and other economically significant illnesses, should be evaluated, and actions should be taken to prevent illegal trade with neighboring countries and the entry of animals without quarantine (42). Our use of molecular epidemiologic techniques provided data suggesting cross-border transmission into Sulaymaniyah governorate, northern Iraq, of PPRV infection circulating in neighboring countries. The viruses we isolated are PPRV lineage 4, which includes viruses whose origins are in the Middle East, Saudi Arabia, and south Asia.  For the control of PPR, attenuated live cell culture vaccines are available commercially. Live attenuating PPRV Nigeria 75/1 isolate vaccine, which belongs to Lineage I (43), is currently used in Iraq. However, current and recent studies in Iraq revealed that Iraqi field strains belonged to Lineage IV, necessitating the development of a specific vaccine by attenuating field isolates of PPRV, and proper administration technique, proper handling, and storage should be followed. The small ruminant populations in the Iraq have not higher protection because of the use of an exotic strain for vaccination in the PPR program (44). Vaccinating ewes during pregnancy with the PPR vaccine is very effective in protecting newborn lambs from the infection (45).
Similar studies are constantly needed in Iraq's diseaseendemic regions to improve disease prevention and control methods and prevent the establishment of new outbreaks of certain virus strains. In order to provide appropriate control measures, national effort must be undertaken to develop an alternative vaccination strain in place of Nigeria/75 that has less variability and better antigenic matching with the lineage IV field strain.

Conclusion
This study provided the valuable information to describe the PPR virus strains that exist in Iraq and provided an explanation for why the virus continues to spread throughout the countryside despite the use of regular vaccination in small ruminant populations; this may be due to the field virus isolate heterologous with the current vaccine strain used for the vaccination program, and the divergence rate is between 8 and 13 percent. The outcome of this study allowed us to create an effective vaccination against the virus strain currently infecting fields, allowing us to control and eradicate the infection.