Confirmation and Sequence analysis of N gene of PPRV in South Xinjiang , China

In China, Peste des petits ruminants (PPR) was officially first reported in 2007. From 2010 until the outbreak of 2013, PPRV infection was not reported. In November 2013, PPRV re-emerged in Xinjiang and rapidly spread to 22 P/A/M (provinces, autonomous regions and municipalities) of China. In the study, suspected PPRV-infected sheep in a breeding farm of South Xinjiang in 2014 were diagnosed and the characteristics of complete sequence of N protein gene of PPRV was analyzed. The sheep showed PPRV-infected signs, such as fever, orinasal secretions increase, dyspnea and diarrhea, with 60% of morbidity and 21.1% of fatality rate. The macroscopic lesions after autopsy and histopathological changes were observed under light microscope including stomatitis, broncho-interstitial pneumonia, catarrhal hemorrhagic enteritis and intracytoplasmic eosinophilic inclusions in multinucleated giantcell in lung. The formalin-fixed mixed tissues samples were positive by nucleic acid extraction and RT-PCR detection. The nucleotide of N protein gene of China/XJNJ/2014 strain was extremely high homology with the China/XJYL/2013 strain, and the highest with PRADESH_95 strain from India in exotic strains. Phylogenetic analysis based on complete sequence of N protein gene of PPRV showed that the China/XJNJ/2014 strain, other strain of 2013-2014 in this study and Tibetan strains all belonged to lineage IV, but the PPRV strains of 2013-2014 in this study and Tibetan strains were in different sub-branches.

More than 70 000 sheep and goats have been culled in September 2014 causing huge loss (Wu et al. 2015).
This study reported the diagnosis of sheep suspected with PPR and the characteristics of the complete sequence of N protein gene of PPRV in a breeding farm of South Xinjiang in 2014.

MATERIALS AND METHODS
There were about 10 000 sheep being raised in a breeding farm in the Akesu region of south Xinjiang.Most of these sheep had been bought from the surrounding townships (indigenous breed, Mekit sheep) and Yili region of Xinjiang (exotic breed), a few of them bought from Gansu province (exotic breed).The scattered sheep which were raised by individual farmers (20-50 sheep per household) combined into a large herd, then rotational grazing.In mid-May of 2014, a herd of sheep with 5 goats was infected after grazing near a reservoir.Five of the dead sheep have been examined using autopsy after on-site diagnosis by staff of the South Xinjiang Diagnostic Center for Livestock and Poultry Disease.The viscus tissues samples of the five sheep were fixed in 10% neutral formalin, separately.
The histopathological changes of the formalin-fixed tissues samples which were conventional paraffinembedded sections and H.E. stained were observed under light microscope.The PPR viral RNA was extracted from the five formalin-fixed mixed tissues samples (lungs, lymph glands, spleens, livers and kidneys) using RNeasy® FFPE kit (QIAGEN, Code No. 217504) in accordance with the instructions provided by the manufacturer.The extracted RNA was used to amplify the DNA by one-step RT-PCR according to the instruction of RNA LA PCRTM Kit (AMV) Ver.1.1 (TaKaRa, Code No. RR012A).The primers of N gene (upstream primer: 5′-AGA GGA GGA GGA GCA AGA-3′ and downstream primer: 5′-GTA TCA GGG TTC GGT GTT-3′) were designed by using Primer PREMIER Version 5.0 software.The optimum annealing temperature was 55℃.The length of amplified products was 1 881 bp.Lyophilized attenuated live PPR vaccine (Nigeria 75/1) bought from Xinjiang Recon Animal Husbandry Bio-Technology was used as the positive control in the study.The PCR amplified products were purified following the instructions of TIANgel Midi Purification Kit (TIANGEN®, Code No. DP209), and sequenced using ABI3730XL sequencer.The SeqMan program and EditSeq program of DNAStar were used to splice and edit the sequences (Table 1), respectively.Identity of the nucleotide sequence and amino acid sequence were reckoned by the Clustal W method of the MegAlign program in the DNAStar software.The phylogenetic tree was reconstructed by the neighbour-joining method of the Kimura twoparameter model in the MEGA5 version 5. The obtained sequences in the study have been submitted to GenBank under the accession numbers "GenBank accession NO.: KX938427" for N gene.The main clinical signs included fever, dullness, diarrhea, lacrimation, matting of eye lids, purulent oculonasal discharges, cutaneous nodules, erosions on the soft palate and gums, and laboured breathing were observed (Fig. 1a).
Color inconsistency in different areas, multiple dark red and firm areas in the lungs were detected (Fig. 1b).Red foamed liquid flowed from section of lungs.The other results of pathological autopsy included haemorrhages and congestion of the intestines, watery intestinal contents (Fig. 1c), uneven section of grey-red lymph nodes, patches of grey-yellow on kidney surfaces (Fig. 1d).There were no apparent macroscopic lesions in other organs.
The histopathological changes of dead sheep suspected with PPR: Lung lesions of bronchointerstitial pneumonia were seen obviously in the sheep.The epithelial cells of the bronchi, bronchioles and alveoli showed hydropic degeneration.The alveoli lumina were filled with edematous fluid, desquamated epithelial cells, erythrocyte and inflammatory cells.The infiltration with larger macrophages and lymphocytes were observed in alveoli and interstitial tissue of the lungs.Hyperplasia of typical syncytial cells both focal and scattered were observed in the alveoli lumina.Intracytoplasmic eosinophilic inclusions in macrophages, syncytial cells and alveolar type II epithelial cells of lung and epithelial cells of the bronchi and bronchioles (Fig. 1e) were observed.The lesions of intestines consisted of widely degeneration and necrosis of epithelial cells of the intestinal mucosa and enteraden, edema, congestion and hemorrhage of the lamina propria mucosae and submucosa which were infiltrated with inflammatory cells mainly consisting of macrophages and lymphocytes.Extensive cellular degenerations and necrosis existed in the parenchyma cells of organs such as liver, spleen, lymph nodes and kidney.In addition, macrophages, lymphocytes and plasmocytes infiltrated in interstitial tissue of the liver, spleen, lymph nodes and kidney were observed.The 45 PPRV strains analyzed in the study were divided into 4 lineages (Ⅰ-Ⅳ) based on the complete sequence of N protein genes and all involved Chinese strains clustered into lineage Ⅳ along with 14 strains from India, 2 strains from Morocco, 1 strain from Ethiopia, and 3 strains from Turkey (Fig. 2).The China/XJNJ/2014 strain and other involved Chinese PPRV strains of 2013-2014 in the study were in same sub-branches, but Tibetan strains from China in 2007-2008 were in other subbranches.Moreover, Phylogenetic analysis revealed that the China/XJNJ/2014 strain was the most closely related to the two Indian strains in 2014.

DISCUSSION
The herd of sheep in the study had PPR symptoms such as fever, orinasal secretions increase, dyspnea and diarrhea.Morbidity and fatality rate of the disease was 60% and 21.1%, respectively.The fatality rate may be above this figure because culled PPRV-infected sheep have not been included.The macroscopic lesions after autopsy and histopathological changes under light microscope included stomatitis, bronchointerstitial pneumonia, catarrhal hemorrhagic enteritis and intracytoplasmic eosinophilic inclusions in multinucleated giantcell of lung.The positive rate of the formalin-fixed mixed tissues samples was 60% by RT-PCR detection.The results of sequencing and sequence analysis of N protein gene were consistent with PPRV gene.It was confirmed that the herd was infected with PPRV when the outbreak occurred.In the study, it should be noted that PPRV were detected from the formalin-fixed tissues samples which have several advantages: to prevent the spread of the virus, do not need cold chain transport and archived pathological tissues samples can be used for retrospective study, without affecting detection of PPRV (Kihu et al. 2015).In 2013-2014, China had suffered the unprecedented outbreak of PPR, including at least 256 counties or banners in 22 P/A/M and over 70 000 sheep and goats have been culled caused huge loss for sheep farming (Wu et al. 2015).Although the presence of PPR was indicated in Tibet as early as 2005 using the descriptive epidemiologic and serologic method and confirmed by molecular epidemiologic detection in Tibet from July through November 2007.Afterwards, three or more outbreaks of PPR were reported between 2008 and 2010 in Tibet.However, from 2010 until the outbreak of 2013, PPRV infection was not officially reported in China (Wang et al., 2009).

Phylogenetic analysis showed that all involved
Chinese strains in the study belonged to lineage Ⅳ, but in different sub-branches with Tibetan strains of 2007-2008 (Bao, et al., 2014, Wu et al., 2015).Outbreaks of PPR have been reported in 7 countries (Mongolia, Russia, Kazakhstan, Tajikistan, Afghanistan, Pakistan and India) adjacent to Xinjiang of China, which means that PPRV was present in these countries.The New Silk Road strategy further drove the development of sheep raised in Xinjiang and led to sheep production increase.Uighurs are overwhelmingly Muslims who have greater need for sheep products in the Xinjiang Uygur Autonomous Region.There were abundant and various wild small ruminant animals in the border of Xinjiang, and PPRV can spread between domestic animals and wild small ruminants, so wild small ruminants in the vicinity of the boundaries of Xinjiang probably played a role in cross-border dissemination of PPRV.In addition, before and after the Chinese New Year, the consumption of mutton was considerable causing frequent sheep transportation to different places.After comprehensive analysis of these reasons, we draw a conclusion that the re-emergence of PPRV in the end of 2013 was probably introduced from neighbouring countries into Xinjiang rather than from Tibet of China.It was reported in the literature that the outbreak of PPR in 2013-2014 in China was probably introduced from Tajikistan and Pakistan (Bao, et al. 2014, Wu et al. 2015).The herd of PPRV-infected sheep in the study belonged to a new sheep breeding farm.Most of sheep in the farm were bought from the surrounding towns (indigenous breed, Mekit sheep) and Yili region of Xinjiang (exotic breed), a few of them bought from Gansu province (exotic breed).Because of the significantly higher price of mutton in South Xinjiang compared to North Xinjiang, sheep and products were transported from North Xinjiang to South Xinjiang.In mid-May 2014, the herd of sheep was infected with PPR after the outbreak of PPR in Yili of North Xinjiang.Considering the above analysis, the outbreak of PPR in South Xinjiang was possibly introduced from North Xinjiang.
Our investigation showed that the sheep in most counties and regions of South Xinjiang suffered a severe pneumoenteritis before outbreaks of PPR in the Yili region of North Xinjiang in 2013.Meanwhile, according to local large veterinary drugs sales departments in South Xinjiang, the records showed that many sheep farmers bought various types of drugs to treat pneumoenteritis in 2013.PPR possibly existed at that time without being recognized, because the disease was yet in the public eye and was misdiagnosed as other pneumoenteritis in small ruminants by local veterinarians.Besides, the transportation vehicles, which may be contaminated by excretions and secretions from infected sheep, were a major risk factor for spread of PPR.Although compulsory immunization with PPR vaccine (Nigeria 75/1) was required by the Ministry of Agriculture of China in all Xinjiang in 2013, some sheep have not developed effective antibodies against PPRV and were susceptible to PPRV for the following possible reasons: (1) The sheep might be missing vaccination because sheep were relatively scattered in large geographical area of South Xinjiang.(2) Some local farmers with backward ideology did not realize the severity of PPR and didn't cooperate in compulsory immunization.
(3) Since some local veterinary departments had poorer facilities and sanitation, some vaccines without proper preservation led to low efficacy.In addition, Tajikistan and Pakistan from which the outbreak of PPR in 2013-2014 in China was probably introduced based on literature are nearer to South Xinjiang than North Xinjiang.According to the above analysis, this outbreak of PPR may be have been earlier in South Xinjiang than that in North Xinjiang.Namely, PPR was likely spread from South Xinjiang to North Xinjiang.The sheep which had received the PPR vaccine in North Xinjiang were less susceptible to PPR.The sheep which were introduced from inland provinces were un-vaccinated and susceptible to PPR, since they were outside the scope of compulsory immunization; however, there were few of these in the herd.All this explains why sheep infected with PPR were mainly indigenous breed.
The spread of PPR from South Xinjiang to North Xinjiang has not clearly been established yet and requires more extensive data to determine.PPRV was introduced from Xinjiang to central-eastern provinces and then spread between inland provinces of China (Wang et al. 2015).Yunnan, Hubei, Anhui, Guizhou and Jiangsu provinces were more severely struck by PPR than Xinjiang although that is where PPR emerged first (Wu et al. 2015).The sheep were relatively less susceptible to PPRV because the compulsory immunization with PPR vaccine was required in all Xinjiang, as early as 2013.However, the unvaccinated sheep were susceptible to PPR in other provinces except Xinjiang.Phylogenetic Arq.Bras.Med. Vet. Zootec., v.69, n.5, p.1105-1113, 2017 analysis revealed that the highest homology of the China/XJNJ/2014 strain shared with the strain from India in September 2014, speculated possible transmission of PPR from China to India.
The number of sheep was about 10 000 around the herd of PPRV-infected sheep in the study, but fortunately, no PPRV-infected sheep were reported or found in the area.It was highly correlated with that the epidemic was under fine control.Because after the outbreak of PPR at the end of 2013, Ministry of Agriculture and relevant authorities of the People's Republic of China highly valued it and taken the following measures (1) forbidding the transportation of sheep and goat in affected area; (2) emergency vaccination of all susceptible animals in threatened area; (3) timely reporting of suspected PPRV-infected animals; and (4) culling of all animals in PPRV-infected herds and on all farms within a radius of 5km.
It should be noted that, according to reports, large ruminates (cattle, buffalo and camel) and Asiatic lion (Panthera leo persica) can also be infected with PPRV besides small ruminates, and morbilliviruses have the inclination to adapt to new host species.Therefore, if PPRV were frequently spread between different host species, this might increase the likelihood of genetic variation of PPRV.Spread of PPR by wild small ruminates should not be ignored and should be closely monitored.Relevant authorities should detect PPRV on host species, region and time by molecular epidemiology to provide a scientific basis for prevention and control of PPR.In addition, the emergence of novel PPRV strains should be monitored in order to avoid it.The vaccine cannot provide protection for animals which were infected with PPRV variants.Currently, 2-3 lineages of PPRV in a region or country have emerged (Maganga et al. 2013) and it was reported that there were lineage Ⅱ and lineage Ⅳ in China (Wang et al. 2015).

Figure 1 .
Figure 1.Post mortem and histopathological changes in sheep with PPR.(a) Gray-white ulceration on tongue.(b) Multiple dark red and firm areas on lung.(c) Red stripe on intestinal mucosa.(d) patches of grey-yellow on kidney surface.(e) Intracytoplasmic eosinophilic inclusions in syncytial cell and alveolar type Ⅱ epithelial cells of lung (H.E.×400).

Figure 2 .
Figure 2. Phylogenetic tree based on the complete sequence of N protein gene constructed, it can show actual time and region sources of PPRV strain.The neighbor-joining method and the Kimura two parameter model were used for phylogenetic analysis in MEGA5 version 5 at bootstrap value 1000 replicates, and values <70% were not shown.Horizontal ines were proportional to sequence distances.Figure indicates the clear division of the four lineages of PPRV.Sequences of south Xinjiang strains were marked with black rhombus (◆). .