Rubella is a deadly virus infection associated with Congenital Rubella Syndrome. Nine recognized genotypes 1B, 1C, 1D, 1E, 1F, 1G, 2A, 2B, 2C and four provisional genotypes 1a, 1h, 1i, 1j are known to circulate all over the world. As there is no baseline data available for rubella genotypes circulating in India, this study was undertaken to standardize rubella RT-PCR and sequencing protocols. Standardization was done following WHO recommended guidelines for rubella molecular surveillance protocols. RT-PCR modifications in WHO protocols like pretreatment of RNA and primer mix and nested PCR was done. Initial standardization was validated by sequencing ATCC rubella virus To-336 strain and R-VAC rubella vaccine-RA27/3 strain. Standardized protocols were used to sequence 739 nucleotide (8731–9469) window region of E1 gene of wild type rubella isolates from Khadakwasla, (Pune) rubella outbreak and clinical samples like throat swabs from rubella outbreak in Himachal Pradesh. Sequence analysis and phylogenetic analysis of wild type rubella isolate (RVi/Pune.IND/92) and clinical sample (RVs/Himachal.IND/49.06) confirmed detection of genotype 2B from India. This was the first attempt in India to initiate rubella genotyping according to WHO recommended guidelines, which will be expanded and will help to study molecular epidemiology of rubella virus.
