Genetic Characterization of a Novel Mutant of Peste Des Petits Ruminants Virus Isolated from Capra ibex in China during 2015

Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR). The spread of PPR often causes severe economic losses. Therefore, special attention should be paid to the surveillance of PPR emergence, spread, and geographic distribution. Here we describe a novel mutant of PPRV China/XJBZ/2015 that was isolated from Capra ibex in Xinjiang province in China 2015. The sequence analysis and phylogenetic assessment indicate that China/XJBZ/2015 belongs to lineage IV, being closely related to China/XJYL/2013 strain. Interestingly, the V protein sequence of China/XJBZ/2015 showed lower homology with other Chinese PPRVs isolated during 2013 to 2014 (94%~95%), whereas it shared 100% identity with three Tibet strains isolated in China 2007. The 3′ UTR, V gene, and C gene were determined to be highly variable. Besides, 29 PPR genomic sequences available in GenBank were analyzed in this study. It is the first time to use PPRV genomic sequences to classify the different lineages which confirmed the lineage clustering of PPRVs using N gene 255 bp fragments and F gene 322 bp fragments. In conclusion, our findings indicate that the PPRVs continue to evolve in China, and some new mutations have emerged.


Introduction
Peste des petits ruminants (PPR) is an important infectious disease of small ruminants [1]. Peste des petits ruminants virus (PPRV) is the etiological agent of PPR belonging to genus Morbillivirus of the family of Paramyxoviridae [2]. PPRV is an enveloped, negative-sense single-stranded RNA virus with the genome of about 15948-15957 nucleotides (nt) in length. The viral genome encodes six structural proteins including nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin (H) and large polymerase (L), and two nonstructural proteins, C and V [3]. of the viruses [7]. PPRVs have been classified into four lineages based on the molecular characteristics of different virus strains (lineages I, II, III, and IV) [8,9]. The strains isolated in Western Africa mainly belong to lineages I and II [5]. Cocirculation of lineage II PPRV along with lineages III and IV is reported in Eastern Africa, and lineage II has been circulating in Central Africa [10]. The PPRVs circulating in the Middle East and Asia are mainly lineage IV strains [5]. More viruses are isolated in different parts of the world and the geographic distribution of PPRVs has become more complicated. In China, most of the strains are included in lineage IV. A 6-nucleotide insertion in the 5 UTR of F gene was found in a China/XJYL/2013 PPR strain in 2013 and a China/BJ/2014 strain in 2014 [11,12]. Several lineage II strains have been also observed in China in recent years [13]. It indicates that the current evolutionary and epidemiological status of PPRVs in China is also complicated. The present study isolated a new PPRV strain from ibex (Capra ibex) in China 2015. The whole-genome was determined, which will contribute to an improved understanding of PPRV epidemiology for the China Himalayan region through wholegenome and sequence analysis from an isolate taken from an infected wild species of caprine.

Clinical Disease.
In the Bazhou region of Xinjiang province, China, 38 ibex died in unusual circumstances from January to February, 2015. It seemed to be PPR according to clinical signs and symptoms including fever, lassitude, anorexia, pneumonia, and diarrhea; the mucopurulent discharge was clearly observed in the eyes and nose of the suspicious ibex, and congested conjunctivae, necrotic lesions on lips and gums were shown. Xinjiang Academy of Animal Science cooperated with China Animal Health and Epidemiology Center to investigate the mortalities in ibex. Lungs, lymph nodes, and swab samples were collected from the suspected ibex, and the postmortem lesion signs were recorded. The postmortem examination revealed congestion in different lobes of lungs. The clinical signs and postmortem examination led to the suspicion of PPR. All the collected samples were kept at −80 ∘ C for the subsequent virus isolation.

Virus Isolation.
The tissue samples were obtained from the infected animal in this outbreak and African green monkey (Vero) cells were used for PPRV isolation. The homogenized tissues were centrifuged for 10 min at 10,000 rpm, 4 ∘ C, to remove the debris. The supernatant was filtered through a 0.22 m membrane. The obtained virus was incubated on previously prepared monolayer Vero cells. The inoculum was removed and replaced with DMEM plus 2% fetal bovine serum after 2 h adsorption at 37 ∘ C. The cytopathic effect (CPE) was observed and recorded every day after virus incubation. The mock-infected Vero cells were used as a negative control. All ethics, field, and experimental work have been approved and performed in compliance with the standard guidelines of the Gansu Animal Experiments Inspectorate and the Gansu Ethical Review Committee (License number SYXK [GAN] 2010-003).

RNA Extraction and PPRV Detection.
The tissue homogenates and prepared PPRV-infected cell culture were used for RNA extraction, the total RNA was extracted with Trizol reagent (Invitrogen) according to the instructions provided by the manufacturer, and the total RNA was resuspended in 25 L of nuclease free water. Five microliters of extracted RNA was used to synthesize the cDNA fragments. The reverse transcriptase-polymerase chain reaction (RT-PCR) for PPRV detection was performed using PPRV partial N gene primers that were reported previously [14,15]. The RT-PCR assay was performed as previously described by Kerur et al. [15]. The amplified PCR products were resolved on a 1.5% agarose gel stained with 0.5 g/mL ethidium bromide.

Sequence
Cloning and Sequencing. The extracted RNA from one collected positive tissue sample was used for cDNA synthesis, and the obtained cDNA were used directly for genome determination using 14 pairs of primers from the previously published article [11]. The amplified fragments were cloned into the pGEM-T easy vector (Promega) for genome sequencing. The termini of the China/XJBZ/2015 were determined by rapid amplification of cDNA ends (RACE) as previously described [16].

Sequence
Analysis. The sequence identity was analyzed using the MegAlign project of the DNAStar software (DNAS-TAR, Inc., USA). The PPRVs genome sequences and partial genes of Chinese strains available in GenBank were collected and used for sequence analysis. The phylogenetic trees were constructed using the clustalx1.83 and MEGA5.1. Phylogenetic analyses were performed using neighbor-joining method and the Kimura-2-parameter nucleotide substitution model in MEGA5.1 (http://www.megasoftware.net/). The numbers of bootstrap replicates were set as 1000.

Virus Replication in Cell
Cultures. The detection of the PPR-infected samples was determined by RT-PCR and virus isolation. The PPR-positive sample generated clear CPE in Vero cells at 48 h after inoculation (hpi), and the infected cells showed more significant cell disruption and partial focal detachment at 60 hpi ( Figure 1). The RT-PCR detection of the virus-incubated cell culture and the CPE results confirmed the successful isolation of this PPR virus. It was the first time to isolate a PPRV strain from ibex in Xinjiang in China. The isolated virus was named as peste des petits ruminants virus China/XJBZ/2015 and maintained at −80 ∘ C.

Genetic Assessment of PPRV.
The nucleotide and amino acid sequences of the different genes of China/XJBZ/2015 were compared with other PPRV reference strains available in GenBank ( Table 2). The results revealed that the nucleotides sequences and amino acid sequences of L, M, and F genes were comparatively conservative, while other regions were divergent (Tables 3 and 4). Of these viral genes, L exhibited the most conservative amino acid identity (95.2%-99.8%) comparing with the listed strains, and M and F showed 94.6%-99.1% and 93.4%-98.7% identity, respectively. The highly variable P, V, and C proteins showed 83.9%-99.8%, 79.5%-100%, and 74.6%-100% amino acid identity, respectively (Table 3). Besides, it was observed that the nucleotide similarity of both the 3 genomic UTR and 5 genomic UTR was variable (88.8%-100% and 85.3%-100%) (

Phylogenetic Tree.
The consensus phylogenetic trees of PPRVs based on the lineage specific 322 bp F gene sequences [8] and the variable region of the N genes (255 bp) [19,20] were constructed to classify the different lineages. Four lineages (lineages I, II, III, and IV) were clearly divided and China/XJBZ/2015 was clustered into lineage IV ( Figure 2). Besides, the phylogenetic trees based on the viral complete genome sequences and V gene sequences were also constructed. The phylogenetic tree based on the complete genome sequences divided the PPRV strains into four lineages as using N and F gene fragments. The phylogenetic tree based on V gene coding region also clearly divided the four different lineages; however, China/XJBZ/2015 was clustered into a different branch comparing with other three clustering methods. It also confirmed the different genetic characteristics of China/XJBZ/2015.

Discussion
PPRV is genetically highly variable like other RNA viruses [5]. Perhaps, the apparent expansion of PPR in North and East Africa and in Asia has occurred despite the existence of an available vaccine [13,14,[21][22][23]. The viral genome and partial fragment sequencing of PPRVs has been widely performed to determine the spread status and to analyse the genetic variability. Despite work on PPR over many years, there are relatively few PPRV sequences available now, and the evolutionary information and spread status of many geographic regions and countries remain unclear due to the limited sequences; therefore, any interpretation of the molecular epidemiology needs to be approached with caution. There are only 29 PPRV genome sequences available in GenBank at the time of the writing of this paper. Therefore, more PPRV genomes information is essential for further research aimed at investigating the distribution of PPRVs and developing strategies for prevention and control of PPR.
In this study, we isolated a PPRV strain from ibex in Xinjiang province in China 2015 by performing CPE assay and viral genome sequencing. The sequence identity analysis results indicated that China/XJBZ/2015 includes some new mutations comparing with previous Chinese PPRVs. It implied that PPRVs have evolved in ibex in China. The alignment of the different genes of the all used Chinese strains indicates that the 3 UTR, V gene, and C gene are the high variable regions. Possibly, these genes can be used as the potential evolutionary markers for analysis of the mutation status of Chinese PPRV strains.  insertion was firstly observed in the 5 UTR of the F gene of China/XJYL/2013in 2013, and after that, almost all of Chinese lineage IV PPRVs include this insertion. It has been reported that F gene is highly conserved between different PPRV strains [14,18]; our results demonstrated the conservative characteristic of F gene in Chinese strains isolated in recent three years. However, eight amino acids in the F protein of China/XJBZ/2015 were different with other Chinese strains isolated from 2013 to 2014. Five of the amino acids are identical with some other reported PPRV strains, and another three amino acid substitutions are new mutations. F protein is believed to disrupt the target cell membrane, hence inducing the virus-cell and cell-cell fusion [24]. The three amino acid substitutions appeared at the residue of 109, 205 and 206 in F protein. Whether these residues are involved in the regulation of fusion process remains unclear [25]. China/XJBZ/2015 resulted in a high mortality to the affected ibex; whether these amino acids substitutions enhanced the attachment or fusion of the virus particle with the ibex cells and increased the pathogenicity should be further studied. High mortality has been observed in Sindh Ibex affected by PPRV in a national park in Pakistan [26]; there was about 38 ibex deaths during the period of 1 month in this study. It indicates PPR may threaten the huge population of ibex. Therefore, more attention should be paid to the surveillance of PPR emergence in ibex.
The analysis of PPRV population variation has been constructed by aligning the partial F gene or N gene [8,20]. In accordance with the previous studies, all the strains were clustered into four different lineages. The phylogenetic trees constructed based on 322 bp F gene and 255 bp N gene are similar to the phylogenetic tree constructed based on the whole-genome sequences. It is the first time to use PPRV genomic sequences to classify the different lineages, and it confirmed the lineage clustering of PPRVs using N gene 255 bp fragments and F gene 322 bp fragments.
In conclusion, a novel mutant of PPRV has been isolated from ibex in China 2015, and the viral genes analysis results suggest that the PPRVs in China continue to evolve. PPR may threaten the huge population of ibex in Xinjiang province in China. Due to the mutation of PPRV, it is desirable to present more virus genome sequences to unravel the evolutionary path, geographic distribution, and spread status in different regions.