Epidemiology and molecular characterization of re-emerged virulent strains of Peste des Petits Ruminants virus among sheep in Kassala State, Eastern Sudan

Background Peste des Petits Ruminants (PPR) is a severe contagious viral disease, which mainly affects small ruminants. PPR is caused by a Morbillivirus that belongs to the family Paramyxoviridae. In this study 12 suspected PPR outbreaks among sheep and goats were investigated in four localities in Kassala State, Eastern Sudan, during 2015—2017. The causative agent was confirmed by a Sandwich Enzyme-Linked Immunosorbent Assay (sELISA), and a Reverse Transcription Polymerase Chain Reaction (RT-PCR) targeting a partial sequence of nucleocapsid protein gene (N- gene) and a partial sequence of fusion protein gene (F- gene). Sequencing and phylogenetic analysis were carried out on six N- gene based RT-PCR products selected from two outbreaks occurred on border and inner localities of Kassala State to determine the circulating lineages of PPRV strains. Identity percentages were determined between isolates in this study and previous Sudanese, and other (African and Asian) isolates which clustered along with them. Results Out of 30 samples, 22 (73.3%) were positive using sandwich ELISA. From 22 s ELISA positive samples, 17 (77.3%) were positive by Ngene based RT-PCR and only 7(43.8%) out of 16 positive samples by N gene based RT-PCR were positive using Fgene based RT-PCR. The sequencing and phylogenetic analysis confirmed involvement of the lineage IV of PPRV in outbreaks among small ruminants in Kassala State and high identity percentage between our isolates and previous Sudanese and other (African and Asian) isolates. Conclusions The present study demonstrates that genetic relationship between PPRV strains circulating in sheep in Kassala State, Eastern Sudan, and PPRV strains characterized as lineage IV in neighboring African countries such as Eretria,Ethiopia, Egypt, and other Asian countries


Background
Peste des Petits Ruminants (PPR) is a severe contagious viral disease which mainly affects small ruminants [1]. There are several reports of PPR occurring in other wild species, particularly in captive wild ungulates [2]. The disease caused by a virus belongs to the genus Morbillivirus of the family Paramyxoviridae [3]  in human, and Canine distemper virus in pets and wild animals species belonging to Canidae, Mustelidae, Procynonidae, Ailuridae, Elephantidae, and large Felidae [4]. PPR is characterized by depression, fever, discharges from eyes and nose, mouth sores, disturbed breathing, cough, diarrhea, and death [5]. PPRV has a single serotype which has been grouped into four distinct lineages were I, II, III, and IV based on sequence comparison of partial sequences of Fgene [6], Ngene [7], or Hgene [8]. All four lineages have been detected in Africa [9]. In Sudan, the first outbreaks of PPR were originally diagnosed as Rinderpest in 1971 and later confirmed to be PPR [10,11], and then outbreaks continued to be reported all over Sudan. The annual vaccination program against PPR in Sudan using an attenuated vaccine (Strain 75/1) has been established in 2002. Since then, a plan to control PPRV was launched, but organized vaccination campaigns were not well performed [12,13].
Kassala State is located in Eastern Sudan and shares porous international boundaries with Eritrea, Ethiopia, and other Sudanese States (Fig. 1). The virus was isolated from infected camels in a severe outbreak in Kassala State during August-October 2004 [14]. In last years, PPR had become endemic, and outbreaks were occurred throughout year involving different species, but virulent strains of the virus had been emerged from time to time causes a huge loss. Also PPR was investigated in four Sudanese states including Northern State, River Nile State, Khartoum State, and Gezira State during 2016-2017 found that the prevalence was higher than previously documented [15]. Antibodies against PPRV were detected in cattle imported from Sudan to Egypt [16]. Prevalence and presence of PPR viral antigen were investigated in pneumonic lungs of apparently healthy camels at Tambul slaughter house, Gezira State, Central Sudan, demonstrating the exposure and probable sub-clinical infection in camels [17].
The present study reported a confirmed molecular characterization of the virulent virus strain among sheep in Kassala State, and its comparison with available sequences from GenBank database provides valuable information about epidemiology of PPR in Sudan.

Outbreaks areas
Kassala State is a border state and major area of animals' exportation. The state is divided into eleven localities. In this study, we have investigated the disease outbreaks in four localities which are rural Kassala, rural West Kassala, rural New Halfa, and Kassala (Fig. 1). The selected localities had records of suspected PPR outbreaks in which some of them are more severe in a period between October, 2015 and February, 2017. In 2015, there was an outbreak in rural new Halfa locality with clinical signs of fever, diarrhea, and abortion. In 2016, outbreaks were located in rural Kassala and rural West Kassala. The main signs were fever, diarrhea, lacrimation and oral erosions. Severe suspected PPR outbreaks among sheep flocks were occurred in February, 2017 in different villages of Kassala State with similar signs. Two of these villages (Noora and Kalhot) were visited after a few hours of onset of the disease symptoms. Noora village was located in rural West Kassala Locality representing the inner site of Kassala State whereas Kalhot village in rural Kassala Locality representing the border site of Kassala State. In these two outbreaks, the animals aged of 6-7 months from Garaj and Gash breeds were affected. Clinical signs are demonstrated in Table 1.
Detailed information about outbreaks from 2015-2017 is presented in Table 1.

Outbreaks data and sample collection
A total of 30 representative samples of 12 outbreaks were collected from sheep and goats with typical clinical signs of PPR in the febrile stage. Nasal, oral, ocular, and rectal swabs were collected using sterile swabs and preserved in transport media until analyzed. Blood in EDTA and fecal samples were also collected. Tissue samples including lymph nodes, lungs, intestines, spleens, and livers were aseptically collected from necropsied sheep. All samples were placed on ice in the laboratory, and then kept at -20 °C until examined. The data of suspected PPR outbreaks are demonstrated in Table 1.

Sandwich ELISA
Sandwich ELISA was performed using a commercially available PPR antigen capture ELISA kit (IDvet, rue Louis Pasteur-Grabels-France). Thirty samples (two goats and 28 sheep) from the 12 PPR suspected outbreaks were screened for the presence of PPRV antigen (nucleoprotein (N)). The test was performed according to the manufacturer's instructions. The reaction optical density was read at 450 nm using a microplate reader (LINEAR, Barcelona, Spain).

RNA extraction
Viral RNA was extracted from 22 samples using a commercial kit Qiamp Mini Kit (Qiagen, Germany) according to manufacturer's instructions.

One step RT-PCR
Twenty two samples which were positive by sELISA were tested for PPRV nucleic acid by one step RT-PCR amplification. The test was carried out using primer set directed to the conserved partial sequence of Ngene:  [19]. RT-PCR for Fgene was carried out at 45 °C for 15 min to activate transcriptase enzyme, 95 °C for15 min as initial denaturation followed by35cycles of 95 °C for 1 min, 50 °C for1 min, and 72 °C for 2 min, and a final extension step was performed at 72 °C for 7 min using Biometra Thermocycler (Analytik Jena, Germany).
PCR products were subjected to electrophoresis in 2% agarose gel stained with ethidium bromide and visualized under ultraviolet (UV) light using a gel documentation system (Analytik Jena, Germany).

Agreement between sELISA and N gene based RT-PCR
The agreement between Sandwich ELISA and Ngene based RT-PCR applied in this study using 12 samples was calculated as concordance percentage (%). Concordance percentage was calculated by division of the number of positive samples agreed by both sELISA and Ngene based RT-PCR by the total number of samples and multiplied by 100.

Sequencing
Six Ngene based RT-PCR products ( three from border site (Kalhot village) and other three from e inner site (Noora village), were sent to Beijing Genomics Institute (BGI) (Beijing, China) for sequencing. The samples were sequenced using Sanger dideoxy Sequencing technology; both forward and reverse sequencing were performed on at least two independently generated PCR products. The sequencing results of six samples were subjected to Nucleotide-Nucleotide BLAST and Nucleotide-Protein BLAST tools in NCBI (www. ncbi. nlm. nih. gov) to confirm affiliation of the sequences to other PPRV isolates and translated sequences to PPRV nucleocapsid protein. Nucleotide sequences were submitted to the GenBank and get accession numbers: MG712792.1, MG712793.1, MG712794.1, MG712795.1, MG712796.1, and MG712797.1.

Phylogenetic analysis
The sequences were retrieved from the GenBank database and used for the construction of a phylogenetic tree using MEGA 6.0 software [20]. The evolutionary history was inferred using the neighbor-joining method [21]. To present the identity relation between Kassala-2017isolates and other isolates from sub cluster1 in constructed tree (Fig. 2), we compared their partial sequences of N gene together. The sequences identities were calculated using MegAlign software (DNASTAR Lasergene, Madison, Wisconsin USA).

Results
Among 30 samples tested by Sandwich ELISA, 22 (73.3%) were positive. Twenty two samples that were initially tested positive by sELISA were tested by Ngene based RT-PCR, and 17/22 samples (77.3%) were positive. Sixteen samples were selected from 17 samples tested positive by Ngene based RT-PCR, 7/16 (43.8%) were positive using Fgene based RT-PCR ( Table 2). The length of the PCR amplicons was 351 bp for Ngene and 448 bp for Fgene. Twelve samples were used to compare sandwich ELISA and RT-PCR (Ngene), and out of them, 11 samples (animals) were positive for ELISA, nine for RT-PCR, and eight for both PCR and ELISA. This revealed an overall agreement (concordance) of 66.7% (8/12) between the two techniques (Table 3). The morbidity, mortality, and case fatality rates of the PPR outbreaks which are investigated in this study were 9.5% (273/2876), 3.2% (92/2876), and 33.7% (92/273) repectively. Sequences' analysis of all six PPRV isolates in this study showed 98.3% -100% identity to each other.

RT-PCR ( +) RT-PCR (-) Total Concordance (%)
Sandwich ELISA ( +) Kurdofan, and River Nile States [22]. In the course of PPR outbreaks surveys, classic clinical signs and symptoms were noticed including fever, diarrhea (bloody), nasal discharge, lacrimation, cough, grinting, oral erosions, salivation, and abortion. Similar manifestations were reported in previous studies in experimentally infected goats by Coucay Hymann et al. [23] and naturally infected goats by Ullah et al. [24]. The morbidity, mortality, and case fatality rates of the PPR outbreaks in this study were 9.5, 3.2%, and 33.7%, respectively. This is in line with Diallo [25] who recorded the morbidity and mortality rates due to PPR ranging from 0 to 90% which are based on local husbandry practices, breed, age, and other risk factors.
In PPR outbreak in Punjab area of Pakistan, the morbidity rate was 31.7% (19/60), mortality rate was 3.3% (2/60), and case fatality rate was 10.5% [26]. In the present study, the presence of PPRV was proved by clinical signs, and antigen detection using Sandwich ELISA and RT-PCR techniques. The overall prevalence of PPRV herein reported was 73.3, 77.3, and 43.8% using Sandwich ELISA, RT-PCRs for Ngene and Fgene respectively. This agrees with Mahajan et al. [27] who reported that NP3 and NP4 primers for Ngene based RT-PCR showed the maximum sensitivity and specificity of PPRV detection in clinical samples compared Fgene primer based RT-PCR. This attributed to the first protein to be produced by PPRV. Ngene transcripts are produced before that of F-gene thus making N gene more suitable target for improving the sensitivity of RT-PCR for detection of PPRV from clinical samples [27]. Upon laboratory investigation, five samples were positive in Sandwich ELISA and were tested negative in Ngene based RT-PCR. This may be attributed to the presence of PCR inhibitors or viral RNA degradation (thermal degradation) in these samples. Phylogenetic analysis of PPRV revealed that lineage I was limited to Western Africa and has not been reported since 1994 whereas, lineage II circulate in Western and central Africa. In addition, lineage III is restricted to East Africa and the Middle East [28]. Molecular typing conducted in this study indicated that Kassala-2017 isolates were clustered with lineage IV of PPRV. This is in accordance with Kwiatek et al. [29] who carried out molecular typing of enormous sampling of PPRV strains in Sudan during 2000-2009 and provided a strong evidence of the Asian lineage IV in Sudan. Similar results were reported by Khalafalla et al. [14] who characterized PPRV strains discovered among camels in Kassala State as lineage IV genotype. Moreover, the current study showed a genetic relationship  [14]. This finding is consistent with Kwiatek et al. [29] who reported that PPRV strains' lineage IV was defined further by two sub-cluster: one included camel and goats' isolates and some of sheep isolates, whereas the other contained only sheep isolates, and a finding suggests a genetic bias according to the host. The current study also indicated genetic relationship between PPRV strains circulating in sheep in Kassala State, Eastern Sudan, and PPRV strains in Eretria and Ethiopia. When comparing nucleotide sequences between Kassala-2017 isolates and other isolates in the sub cluster1, it is found that there is high identity with these isolates that reach 100% (Fig. 3 and Fig. 4). These close similarities between Kassala-2017 isolates and Eretria and Ethiopia isolates ensure transboundary circulation of PPRV due to free movement of animals searching for pastures across porous borders between Kassala, Eritrea, and Ethiopia. In addition there are live animals trading of camels and small ruminants for meat consumption between Kassala and Egypt through (Shalatain) aperture (Northern border of Sudan) in Red Sea State that it may be the main factor of similarity of viruses circulating in Kassala and Egypt. In addition there is a major trading activity between Saudi Arabia and Kassala State through airlines or through Swakin port on Red Sea..

Conclusions
It could be concluded that PPRV belongs to the lineage IV genotype, which is responsible for outbreaks among sheep in Kassala State. In addition, the existence of the lineage IV in Kassala State may be due to spread of this lineage through transhumance and cross-border trade in small ruminants from neighboring countries where lineage IV is reported. Therefore, regular vaccination and regulation of animal movement between countries are important to establish crucial eradication program of PPR in Sudan.