Peste des Petits Ruminants Virus in Tibet, China

Serologic and molecular evidence indicates that peste des petits ruminants virus (PPRV) infection has emerged in goats and sheep in the Ngari region of southwestern Tibet, People’s Republic of China. Phylogenetic analysis confirms that the PPRV strain from Tibet is classified as lineage 4 and is closely related to viruses currently circulating in neighboring countries of southern Asia.

Serologic and molecular evidence indicates that peste des petits ruminants virus (PPRV) infection has emerged in goats and sheep in the Ngari region of southwestern Tibet, People's Republic of China. Phylogenetic analysis confi rms that the PPRV strain from Tibet is classifi ed as lineage 4 and is closely related to viruses currently circulating in neighboring countries of southern Asia. P este des petits ruminants (PPR) is an acute and highly contagious viral disease contracted by small ruminants such as goats and sheep and causing high rates of illness and death. The disease is endemic in parts of sub-Saharan Africa, the Middle East, and Asia. The PPR virus (PPRV) genogroup consists of 4 lineages (1,2). PPRV infection was offi cially reported in the Ngari region of western Tibet, People's Republic of China, in July 2007. Our study assesses the prevalence of PPRV infection in goats and sheep by region in Tibet. We also characterize strains of the virus by comparing part of the genome sequences with other PPRV sequences available in the GenBank database.

The Study
Small ruminants in regions throughout Tibet were examined for PPRV antibody from July 2007 through November 2007. The sampling procedure focused on 3 groups of animals. The fi rst comprised 718 animals in 4 counties (Rutog, Ge'gyai, Gerze, and Zada) in the Ngari region, where animals having clinical signs of PPRV infection had been reported by local authorities. The second group included 298 animals in Gar and Bulang counties in the same region and in 2 counties bordering the Ngari region (Nyima in Nagqu region and Zhongba in Shigatse region). The third group contained 520 animals in 5 counties within 3 separate regions (Nyalam and Yadong in Shigatse region, Cona and Lhozhag in Shannan region, and Zayu in Nyingchi region).
Field samples, including organ (lymph node, spleen, lung, and intestine) and swab specimens, were obtained from 49 goats and sheep suspected of being infected with PPRV. These animals inhabited 4 counties in the Ngari region (Ge'gyai n = 33, Zada n = 7, Gerze n = 5, and Rutog n = 4). Two reverse transcription-PCRs (RT-PCR) and 1 newly developed and validated real-time quantitative RT-PCR (qRT-PCR) were conducted to determine whether the animals had viral RNA (4-6).  23), indicating high viral loads. However, no animal from Zada County had a Ct <30 (Figure 1). The study confi rmed 11 outbreaks in 4 counties in the southwest Ngari region in Tibet. Nine of the 11 occurred in southern Rutog County, northern Ge'gyai County, and Gerze County, and samples from these 9 were seropositive for PPRV (Figure 1, panel A). By using qRT-PCR and RT-PCR, we found that samples from 4 of these outbreaks were also seropositive for PPRV virus RNA. This fi nding, confi rmed by sequencing, indicates that the main portal of entry for the disease is through southwestern Rutog County. Viral RNA was also detected and confi rmed by sequencing in 1 outbreak in southern Ge'gyai County and another outbreak in Zada County (Figure 1).
The nucleic acid sequences obtained from the PCR products were aligned with sequences from PPRV strains available in GenBank. Partial sequencing (448 bp) of the F gene showed that 20 of 21 samples were identical over the portion of the genome that was characterized (Gen-Bank accession no. EU816772). One (GenBank accession no. EU815053) differed from other Ngari sequences by 1 nt. The Ngari sequences showed a level of nucleotide identity with other PPRV strains of 88.8%-98.8%. Strains of PPRV from Tibet were classifi ed as lineage 4 and were closely related to the India/Bsk/Guj/05 strain isolated in India in 2005 (Figure 2, panel A). Partial Ngene sequences (351 bp) from 18 of 19 Ngari samples were identical (GenBank accession no. EU068731), and 1 sequence (GenBank accession no. EU340363) differed by 1 nt. Sequence comparison of the Ngari N gene to the sequences of other PPRV strains showed a nucleotide identity level of 81.6%-97.3%. Strains of PPRV from Tibet were classifi ed as lineage IV and were closely related to the Tajikistan/04 isolate found in Tajikistan in 2004. Different kinds of numbering were used for the phylogenetic comparisons for the 4 lineages of these 2 genes. The lineages were classifi ed as 1-4 in F gene analyses (1). Later research classifi ed the lineages in N gene analyses as I-IV (2) (Figure 2, panel B).

Conclusions
In this study, PPRV was found by collecting samples from animals in the fi eld and detecting infection by using competitive ELISA and RT-PCR. Our research provides valuable data on PPRV infection in small ruminants in Tibet. Infection was observed in 4 counties in the Ngari region of southwestern Tibet. Most outbreaks occurred in Rutog and Ge'gyai counties; 1 outbreak was confi rmed in Gerze County and another in Zada County. Epidemiologic and serologic evidence suggests that the infection fi rst emerged in Rejiao village in southwestern Rutog from No-  Also, this disease is frequently confused with other diseases that cause respiratory problems and death in small ruminants (7). The molecular epidemiologic techniques provided data suggesting cross-border transmission of PPRV infection into Tibet. PPR infection has been recognized in many Asian countries bordering southwestern China, including India (8), Nepal (9), Bangladesh (9), Pakistan (10), and Afghanistan (7). Almost all recent viruses from southwest Asia and the Middle East belong to PPRV lineage 4. The virus that circulated in the Ngari region is of the same lineage and is closely related to an isolate from India (2005) and an isolate from Tajikistan (2004). Close contact between susceptible animals and infected animals in the febrile stage is the main method of transmitting PPR. The terrain of western and southwestern Ngari permits uncontrolled animal movement, and a small ruminant trade exists between Tibet and bordering nations such as India and Nepal. These factors and the history of PPRV in Asia suggest that animals from a neighboring country in southwest Asia are likely sources of this infection in Tibet.
Dr Zhiliang Wang is the director of the National Diagnostic Center for Exotic Animal Diseases in the China Animal Health and Epidemiology Center, Qingdao. His main research interests are emerging infectious animal diseases, especially bovine spongiform encephalopathy, scrapie, and peste des petits ruminants. PPRV strains sequenced in this study are highlighted in gray. Other sequences are from GenBank. Phylogenetic analyses were completed with MEGA 3.1 software that used a neighborjoining algorithm and absolute distances and that followed 1,000 bootstrap replicates. The RBOK vaccine strain of rinderpest virus was included as an outgroup. The tree is based on the partial sequence of the fusion (F) protein gene (A) and the nucleocapsid (N) protein gene (B). Different classifi cations were used for the phylogenetic comparisons for the West African lineages 1 and 2. Nigeria and related strains have been classifi ed as lineage 1; the Côte d'Ivoire and related strains have been classifi ed as lineage 2 (1). Later research reversed this order in classifying the lineages in N gene analyses (2).