Peste des petits ruminants (PPR) is an acute, highly contagious, notifiable and economically important transboundary viral disease of sheep and goats. In this study, sequence and phylogenetic analyses of structural protein genes, namely the nucleocapsid (N), the matrix (M), the fusion (F) and the haemagglutinin (H) coding sequences of virulent and vaccine strains of PPR virus (PPRV), were undertaken to determine the genetic variations between field isolates and vaccine strains. The open reading frame (ORF) of these genes of the isolates/strains was amplified by RT-PCR, cloned and sequenced. The ORF of N, M, F and H genes was 1578, 1008, 1641 and 1830 nucleotides (nt) in length and encodes polypeptides of 525, 335, 546 and 609 amino acids (aa), respectively, as reported earlier. Comparative sequence analyses of these four genes of isolates/strains were carried out with published sequences. It revealed an identity of 97.7-100% and 97.7-99.8% among the Asian lineage IV and 89.6-98.7% and 89.8-98.9% with other lineages of PPRV at nt and aa levels, respectively. The phylogenetic analyses of these isolates based on the aa sequences showed that all the viruses belonged to lineage IV along with other Asian isolates. This is in agreement with earlier observations that only PPRV lineage IV is in circulation in India since the disease was first reported. Further, sequence analysis of the thermostable/thermo-adapted vaccine strains showed no significant changes in the functional or structural surface protein-coding gene sequences. It is important to monitor the circulation of the PPRV in susceptible animals by H gene-based sequence comparisons in addition to the F gene- and N gene-based approaches to identify the distribution and spread of virus in the regular outbreaks that occur in endemic countries like India.

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Peste des petits ruminants (PPR) is a contagious, notifiable and economically important transboundary viral disease of small ruminants. In this study, a hydrolysis probe-based real-time reverse transcription-polymerase chain reaction (rt RT-PCR) assay for the detection and semi-quantification of PPR virus (PPRV) nucleic acid was developed using the virus RNA and matrix (M) gene-specific primers with Hex-labelled fluorescent probe and applied for the detection of PPRV in clinical samples to identify outbreaks and to monitor the prevalence of disease. The assay was found specific with a sensitivity detection limit of 0.5 pg of total PPRV RNA. Based on a serial dilution of the live-attenuated PPR vaccine virus, the detection limits were approximately 0.1 and 1 TCID₅₀ for the hydrolysis probe and conventional RT-PCR assays, respectively. The assay was linear within a range of 50 ng to 0.5 pg total virus RNA with an intra-assay coefficient of variation (CV) in the range of 0.91-2.86% and an inter-assay CV ranging between 0.59% and 2.37%. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the experimental/field clinical samples. This assay detected the PPRV in pre-clinical swab materials as early as the 4th day post-infection (dpi) and up to 17th dpi in nasal, ocular and oral swabs collected from experimentally infected animals. The rt RT-PCR was rapid, specific and 10 times more sensitive than conventional RT-PCR. It is an alternative test to the existing diagnostic assays and could be useful with enhanced applicability in field clinical diagnosis by avoiding the use of expensive commercial real-time PCR reagents. This assay was adopted directly in the detection of PPRV nucleic acid in clinical samples collected from sheep and goats suspected of PPR to monitor outbreak situations and the clinical prevalence of PPR in India.

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Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.