Molecular epidemiology of peste des petits ruminants viruses from southern India

PESTE des petits ruminants (PPR) is a highly contagious disease affecting sheep and goats which is widely distributed in sub-Saharan Africa and the Arabian Peninsula (Taylor 1984). The virus is antigenically related to rinderpest virus, which infects cattle and other large ruminants. It is a member of the morbillivirus genus, which also includes measles, canine distemper virus and viruses of marine mammals, in the family Paramyxoviridae. In India, PPR virus was first identified in an outbreak in 1987 (Shaila and others 1989) and since then it has been reported in all parts of the country (Nanda and others 1996). It is now assumed that PPR was probably present in India before 1987 but was confused with rinderpest (Taylor and others 2002). In order to obtain epidemiological information on the nature of the viruses involved in PPR outbreaks, the genetic relationships between the viruses from different geographical regions of the world have been determined by sequence comparisons of a segment of the virus fusion (F) protein gene, by Shaila and others (1996). In that study, a phylogenetic tree was constructed using several different PPR viruses and it was found that there were four different lineages. PPR viruses from Africa were found to group into three distinct lineages. Viruses isolated from Africa in the early 1970s formed one lineage (group 1), those isolated from the Ivory Coast and Guinea in the late 1980s formed a second lineage (group 2) and the third group included viruses isolated from Sudan, Oman and southern India over a 20-year period. The more recent outbreaks, which occurred across Asia (including northern India) in the 1990s, fell into a fourth lineage (group 4). The existence of distinct lineages of viruses in northern and southern India calls for a more detailed molecular analysis of PPR viruses involved in later outbreaks. The present study was undertaken to confirm the present situation with respect to the molecular epidemiology of the PPR viruses circulating in southern India since 1996. Seven PPR virus isolates were used in this study, six from Tamil Nadu and one from Andhra Pradesh, two states of southern India. The place and year of isolation and the nature of the sample used for RNA isolation are given in Table 1. Viruses were grown on Vero cells maintained in minimum essential medium supplemented with antibiotics and fetal calf serum. Infected cells were harvested when extensive cytopathic effects were seen. RNA was isolated from infected Vero cell monolayers, from tissues obtained from dead animals at postmortem examination or from oculonasal swabs from live animals using the method described by Chomczynski and Sacchi (1987). The total RNA extracted was reverse transcribed into CDNA using the Thermoscript reverse-transcriptase PCR kit (Life Technologies), following the method of Forsyth and Barrett (1995). For the PCR, two sets of F gene-specific primers were used. Primers F 1 and F2 amplified a 372 base-pair product, while primers FIA and F2A amplified a 308 base-pair product, in a nested reaction. The sequences of the primers are described by Forsyth and Barrett (1995). The PCR was performed in a thermal cycler (MJ Research) using the Taq PCR core kit (Qiagen) and the following programme: step 1, one cycle, 94°C for five minutes; step 2, 35 cycles, 94°C for one minute, 55°C for one minute, 72°C for one minute; step 3, one cycle, 720C for seven minutes. The amplified PCR products of the first reaction were confirmed in the nested reaction. After confirmation of the product as part of the PPR virus genome, the outer PCR product (produced by primers Fl and F2) was gel purified using a DNA extraction kit (Fermentas). The purified PCR products were sequenced using the automated ABI prism module at Bangalore Genei, Bangalore, India, using cycle sequencing methods. The nucleotide sequences were aligned with each other using the ClustalV programme and the phylogenetic analysis was carried out using molecular evolutionary genetic analysis (MEGA) software, version 1.02 (Sudhir and others 1993). The parameters used were Universal genetic code, Jukes Cantor distance and UPGMA. These partial F gene sequences were compared with the already available PPR F sequences and an updated phylogenetic tree was generated. When the nucleotide sequences of the seven isolates from southern India were aligned along the sequence available for one other isolate from southern India (Ind TN 92/1) (Shaila and others 1996) it was seen that all of the isolates collected from 1996 to 2001 belonged to the same group, and differed from the Ind TN 92/1 isolate (Fig 1). The first isolate of PPR virus from India, from an outbreak in 1987 and named as Ind TN 88/1 (Shaila and others 1989) also grouped with the six more recent virus isolates. Another candidate vaccine virus Ind TN 97, subgrouped with the Ind TN 88/1 isolate, but was slightly different from the others. However, when the amino acid sequences coded by the fragment of the F gene amplified by PCR were compared among these isolates, no differences were seen. Thus, it appears that only the Ind TN 92/1 isolate had different nucleotide sequences, aligning itselfwith Middle Eastern virus isolates, while the other viruses from southern India did not. In a previous study (Shaila and others 1996), it was also reported that a unique epidemiological situation was present