There are few reports on circulating wild type measles genotypes in India. In 2002, circulation of measles genotype D4, D8 and A was reported from Pune, Maharashtra . The circulation of genotypes D4, D7 and D8 in some locations in Tamil Nadu has been reported [15, 16]. There were no reports available on molecular characterization of measles virus from Uttar Pradesh a state of North India. Therefore, this study was conducted in Uttar Pradesh to establish a baseline molecular data about the circulating measles virus genotypes. Since the genetic diversity of measles viruses circulating in a state could be correlated with its immunization coverage, which varies between the states in India, it is essential to establish statewide molecular data of measles viruses.
In the present study 38 strains were obtained from 15 villages of 12 districts. All the strains were obtained from the villages. The studied population in the present study comprised of 3507 children. The known vaccination status of studied population was only 28%. Among the 526 cases investigated, 26 (4.9%) cases were immunized (as evidenced by availability of immunization card or statement of parents/ health workers). Immunization status was not known for 500 (95%) cases or not able to recall vaccination history. More males were vaccinated compared to females in the present study. In the present study the majority of cases were of lower socioeconomic status, illiterate and less aware of vaccination programs. A significant high severity of symptoms has been found in another study in cases belonging to the lower socioeconomic status , especially among illiterates; an indirect indicator of poor hygiene and awareness for the vaccination program.
In our study, we also found that sex, maternal literacy, social category, maternal occupation, and standard of living were important “demand-side” predictors in the immunization status of children, which was found in other studies as well [19–21]. While some studies  have shown a significant role of health workers in reducing sex bias, we found that, despite adjusting for the role of health services and presence of health workers, girls are less likely to be immunized than boys.
A higher proportion of males were affected in the present study as compared to the females which is corroborated by few other studies [23, 24]. The recruitment bias was minimized due to house to house survey. The possible reason for more males get infected could be the differential attitude of parents towards female child or gender difference in the outbreak area.
All the blood samples were collected between 4 and 15 days of rash onset. The blood samples were collected from clinically suspected measles outbreaks (based on the WHO case definition). From the results of measles IgM positivity based on the timing of sample collection, it was noticed that 227 samples (97%) and 8 samples (3%) that were collected between days 4 and 15 after the onset of rash were positive and equivocal respectively. The timing of sample collection is probably responsible for negativity in epidemics. The optimum time for blood sample collection for IgM detection is 4–28 days post onset of rash [25, 26]. This was also confirmed in the present study. We also noticed such finding by other Indian researchers where it is well documented about 100% positivity for IgM ELISA where blood samples were collected between days 4 and 22 after the onset of rash, where as samples collected between days 1 and 3, 17% positivity were noticed .
Virus isolation from urine sample attempted in the Vero/hSlam cell line yielded virus from 20 samples (15%). Similar low success rates were observed in previous studies, were 12%  and 18%  yields were found. RT-PCR analysis shows only 28% (39 samples) positivity.
Reason for low yield of virus isolation from cell culture and RT-PCR could be timing of sample collection and transportation of the samples to the laboratory. Previous finding clearly demonstrated that percentage of virus isolation varied based on timing of sample collection. The urine samples collected between 8–13 days shows 33% positivity and sample collected between 14 to 20 days shows 29% positivity . All the positive samples reported in the present study by cell culture and RT-PCR analysis was collected within 6 days of rash onset. All the samples collected beyond 6 days were negative.
The percentage of virus isolation also varied based on cell culture and RT-PCR analysis. The high percentage positivity by RT-PCR as against virus isolation was also reported previously from India [14, 15]. Study from Suburban Khartoum  also showed high percentage positivity by RT-PCR compared to virus isolation.
All these findings indicate the fact that the optimum concentration of virus and timing of collection of sample is required for the isolation of the virus and RT-PCR is more sensitive as compared to virus isolation.
Optimally, measles virus is excreted from infected cases only for the first 5–7 days after rash onset, often in low titers. WHO has recommended samples for virus isolation should be collected within 5 days after rash onset . For these reasons, attempts to detect virus from suspected measles cases after a week of rash onset is not considered to be a useful diagnostic tool.
It is well understood that measles case confirmation by virus isolation is less sensitive. The measles virus genome is relatively stable and shows minor detectable changes over the course of an outbreak or even over 12 months. Hence, isolation of virus from all cases is not considered necessary and 1 or 2 isolates from each outbreak or chain of infection will provide sufficient data to determine transmission pathways .
During the study period genotype D8 found to be circulating in Uttar Pradesh India. All the cases reported in the present study did not have travel history or contact with traveler, suggesting that these viruses are indigenous.
The genotype D8 detected in the present study was differed from each other by maximum of 3.4% while some strains were 0.2% differ from each other. Genetic heterogeneity of the Indian measles viruses is not a result of increased mutation rates but is due to the presence of multiple co-circulating lineages of viruses within the endemic region.
This analysis clearly demonstrated that multiple lineages of genotype D8 are co-circulating and disseminated widely throughout the state, a pattern consistent with an endemic genotype.
Though measles surveillance in India is in its infancy, during the preceding 15 years only Clade D genotypes (D4, D7, D8) have been detected in India, whereas surrounding countries have detected D4, D7, D8, D9, D5, H1, d11, and G3 genotypes [9, 31]. These external genotypes were not imported into India between 1995 and 2010. The possibilities of missing genotypes or of importation of other genotypes must be further studied and documented with continuous countrywide molecular surveillance in India. Other countries have also documented co-circulation of genotypes, probably due to multiple chains of transmission during an outbreak [32, 33].
The mutation rate amongst field isolates of measles virus is low and appears to be random rather than driven by vaccine pressure or immune responses. Within a genotype, nucleotide difference (virus lineage) can assist in distinguishing separate episodes of transmission . In the present study the nucleotide difference was observed based on the outbreak / transmission chain. In countries or regions with endemic measles, many lineages of a single genotype may co- exist; however as countries begin to move from endemic to epidemic measles the diversity of sequences within the circulating genotypes decreases [35–38]. This is consistent with the present study also; multiple lineages of genotype D8 strains were circulating in the state during 2008 – 2011. However, in 2002, Oliveira MI et al. have reported, the genotype D6 virus associated with a large measles outbreak that occurred in several South American countries between 1996 and 1997 had identical N gene sequences suggesting rapid spread of a single lineage . Analysis of measles viruses circulating in Burkina Faso, before and after a mass vaccination campaign, showed that the number of circulating lineages was greatly reduced following the campaign. Sequence analysis of viruses isolated from outbreaks that occurred after the vaccination campaign suggested that virus was introduced from a single source .