Genetic analysis of peste des petits ruminants virus from Pakistan
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Peste des petits ruminants (PPR) is an endemic and highly contagious disease in small ruminants of Pakistan. Despite the fact that an effective vaccine is available, outbreaks are regularly occurring in the country. Thus so far, the diagnosis has primarily been made based on clinical outcome or serology. This study was carried out to characterize PPRV from an emerging wave of outbreaks from Punjab, Pakistan.
A total of 32 blood samples from five different flocks were tested with real-time PCR for the presence of PPRV genome. The samples detected positive in real-time PCR (n = 17) were subjected to conventional PCR for the amplification of the nucleoprotein (N) gene. Phylogenetic analysis of the sequenced N genes (n = 8) indicated the grouping of all the sequences in lineage IV along with PPRV strains from Asian and Middle East. However, interestingly sequences were divided into two groups. One group of viruses (n = 7) clustered with previously characterized Pakistani isolates whereas one strain of PPRV was distinct and clustered with Saudi Arabian and Iranian strains of PPRV.
Results demonstrated in this study expanded the information on the genetic nature of different PPRV population circulating in small ruminants. Such information is essential to understand genetic nature of PPRV strains throughout the country. Proper understanding of these viruses will help to devise control strategies in PPRV endemic countries such as Pakistan.
KeywordsSmall Ruminant Genetic Nature Contagious Viral Disease Good Control Strategy Routine Diagnostic Purpose
Peste des petits ruminants
Polymerase chain reaction
Competitive enzyme linked immunosorbant assay.
Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild ruminants where it cause high morbidity (100%) and mortality (90%) [1, 2]. The causative agent, peste des petis ruminants virus (PPRV), is grouped in the genus Morbillivirus within family Paramyxoviridae along with rinderpest, measles, phocine-, dolphin-, canine- and porpoise-distemper viruses . Like other Morbilliviruses, PPRV is pleomorphic, with negative sense single stranded RNA genome containing 15,948 nucleotides [2, 4]. The genome follows the “rule-of-six” and encodes six structural proteins that include nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin (H) and large polymerase (L) .
Based on the molecular characterization, strains of PPRV can be grouped into four lineages, which are genetically distinct from each other. Lineage I includes isolates from Western Africa, lineage II contains isolates from West African countries, the Ivory Coast, Guinea and Burkina Faso, and lineage III represents strains from Eastern Africa, the Sudan, Yemen and Oman [2, 4, 5, 6]. The lineage IV comprises of PPRV strains from the Arabian Peninsula, the Middle East and South Asia [2, 4, 6]. Classification of PPRV is being analyzed based on the sequence analysis of both F and N genes; however, parallel comparison of PPRV strains has proposed that N gene is most divergent and hence most appropriate for molecular characterization of closely related isolates . The virus has been recognized to occur as only one serotype among four lineages .
PPRV has been documented in Pakistan since 1991, however, the isolates were confirmed through PCR few years latter in 1994 . Since then, PPR remained endemic in Pakistan despite use of a live attenuated vaccine in small ruminants. Due to serological monitoring facilities, it remained difficult to ascertain the level of vaccine failure and thus aggravates disease epidemiology and its control. Therefore, determining the nature of circulating strains in different parts of Pakistan is crucial to not only aid in disease diagnosis and but also to devise better control strategies in future. The present work has been conducted to determine the genetic nature of circulating PPRV strains that are continuously causing outbreaks in central Punjab, Pakistan.
Brief history of outbreaks and outcome of different diagnostic assays
Place of sampling in district Okara
Animals sampled/total animals in the herd
Real-time PCR (positive/total)
Conventional PCR for N gene (positive/total)
High fever (105–107 F), nasal discharges, sneezing, coughing, sticking of mucus to nostrils, erosions in the oral mucosa. Severe diarrhea and then drop in temperature after start of the diarrhea. Death nearly 5–10 days after start of disease
Fever (104-105 F), nasal discharges, sneezing, coughing, sticking of mucus to nostrils, erosive ulcerative stomatitis, diarrhea
Fever (104-105 F), nasal discharge, sneezing, coughing, necrotic stomatitis, severe diarrhea
Body temperature (105–107 F), weakness, nasal and lacrimal discharges, sneezing, coughing, erosive lesions in the oral cavity, severe diarrhea
Fever (104-105 F), purulent nasal discharges, coughing, erosive ulcerative stomatitis, diarrhea
The total RNA was eluted from the QIAcard FTA Indicator as described  and was used to screen by real-time PCR for the presence of PPRV genome as reported earlier [9, 10]. All the samples appear positive in real-time PCR (Ct values <35) were used in conventional PCR to amplify N gene of PPRV for downstream sequencing [9, 11]. The resultant PCR products were gel extracted and processed for sequencing using ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems), according to the manufacturer’s instructions. Sequences were analyzed with an automated nucleic acid analyzer (ABI PRISM 3100; Applied Biosystems). Each DNA fragment was sequenced at least twice in both directions.
The sequences were edited and assembled using EditSeq and SeqMan suits within Lasergene 8 (version 8.0.2 13, DNASTAR, Inc., Madison, WI, USA). The resultant sequences were aligned with the sequences retrieved from GenBank representing all the lineages of PPRV. Construction of phylogenetic trees was performed with the neighbour-joining method using Kimura two-parametermodel in Mega5 version 5 (CEMI, Tempe, AZ, USA).
Out of total samples (n = 32) analyzed, real-time PCR detected only 18 samples with Ct values lower than 35. The results of real-time PCR confirmed the cause of these outbreaks to be PPRV. However, to demonstrate the genetic nature of PPRV, a conventional PCR for the amplification of N gene was performed. Although all the samples were not detected positive, a representative sample of each outbreak was amplified which appear sufficient for sequencing and downstream analysis. All the N genes were submitted to the GenBank under accession number KC207867-KC207874.
Since first report of PPRV in Pakistan, several outbreaks have been reported [2, 9, 12, 13, 14, 15, 16, 17, 18, 19]. Most of these were based on either clinical history, antigen or antibody detection. However, molecular characterization of the virus is considered a key to understand the genetics of the causative agents. The results of real-time and conventional PCR from blood samples, transported onto FTA cards, confirmed the fact that they are suitable, reliable and economical mean of transportation [9, 19], especially where maintenance of cold chain is not possible or legislations for shipment of biological material is complicated . Conventional and real-time PCR for the detection of PPRV have shown variable sensitivity and specificity. Therefore these tests must be evaluated in parallel before their implementations for routine diagnostic purposes in specific laboratory settings.
In conclusion, lineage IV of PPRV is currently circulating in the country, with certain level of genetic diversity. Since serological assays (i.e. cELISA) don’t necessary indicate the current persistence of the infection, it is essential to implement molecular diagnosis along with characterization. Despite PPRV is endemic in Pakistan and outbreaks are regularly occurring, limited information is available on the genetic nature of PPRV. The sequences reported here expand the available information on the circulating strains of PPRV in Pakistan. There is no sequence information for any of the N genes of PPRV is available other than our previously characterized sequences and the sequences reported here. With these limited data, we still manage to demonstrate the presence of two different population of PPRV, which warrant future need to perform such studies at country level to ascertain the complete picture of circulating viruses in the country. Such understating is crucial for devising future control plans. Currently, vaccination is recommended in certain areas of the country. This vaccination is based on Nig75/1, which belong to lineage II, while field isolates from Pakistan are grouped in lineage IV. Genetic characterization of field strains will provide foundations for construction of vaccines from domestic strains as has recently been practiced in India.
Authors would like to thank Jenna Anderson for critical reviewing of the manuscript.
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