3.2. Genotyping analysis
Among 61 HAdV PCR-positive specimens, 57 were genotyped using penton gene-specific primers. Four non-typable specimens (Ahmadabad n=2; Surat n=2) were confirmed to be HAdV after nucleotide sequencing of the partial hexon gene. Analysis of the nucleotide sequence data of the HAdV strains was carried out using the BLAST and phylogenetic analysis. The phylogenetic analysis of the study strains depicts the grouping of the study strains in different subgroups and corresponding genotypes with high bootstrap support (Figure 1). Among 61 HAdV positive specimens, 52.4% (n=32) belong to HAdV-F and genotyped as HAdV-40 (n=14) and HAdV-41(n=18). The HAdV-40 and HAdV-41 strains of the study clustered with corresponding reference strains (MK883611; MH465394) and showed 100 percent nucleotide identity (PNI) values. Eleven strains (17.4%) grouped with subgroup HAdV-A, and genotyped as HAdV-12 (n=4), HAdV-18 (n=2), and HAdV-31 (n=5) and showed 98-100, 100, 99.5, and 99.3-100 PNI values with corresponding prototype strains. Subgroup HAdV-B with genotype HAdV-7 observed in five (8.2%) specimens with 99.4 - 100 PNI values with MNO11575.1 prototype strain. Seven study strains (11.4%) were classified as subgroup HAdV-C and genotyped as HAdV-2 (n=5) and HAdV-5 (n=2) with 94.9- 100 and 97.8- 100 PNI values with corresponding prototype strains namely JX173081 (Egypt) and KF429754 (USA) respectively. Subgroup HAdV-D observed in two specimens with PNI value of 98.6 with HAdV-23 (Prototype KF268327,), and 98.7 with HAdV-69 (Prototype KJ626292) strain.
The distribution of the different HAdV subgroups and genotypes in different study years and cities is shown in Table 3. The subgroup F strains were detected in all four cities with 11, 8, 8 and 5 strains in Ahmadabad, Mumbai, Surat and Pune receptively. It should be noted that the HAdV-7 strains of subgroup B were found only in Pune city with absence of subgroup A in Pune and subgroup C strains in Surat. Two strains of subgroup D identified in the study were isolated from AGE patients of Pune and Surat city.
Analysis of the distribution of the HAdV-F strains showed 30.0 (95% C.I: 1.60, 58.40), 65.2 (95% C.I: 45.75, 84.68), 60.0 (95% C.I: 35.21, 84.79) and 55.6 (95% C.I: 23.09, 88.02) percent in the year 2013, 2014, 2015 and 2016. Similar analysis for cities namely Pune, Mumbai, Ahmadabad and Surat, showed 31.2 (95% C.I 8.54, 53.96), 61.5 (95% C.I 35.09,87.99), 78.6 (95% C.I 57.08,100.00) and 57.1 (95% C.I 31.22,83.07) percent patients with HAdV-F infections respectively. The difference in percent positivity among different years as well as cities is not statistically significant with exception of the significantly low proportion of HAdV-F cases in Pune compared to Ahmadabad (p = 0.0097). HAdV-F strains were identified consecutively between 2013-2016, 2014–2016 and 2013-2015 in Ahmadabad, Surat and Mumbai respectively and in Pune only in 2013 and 2015.
Comparison of the mean age of the patients infected with different subgroups (A, B, C, D and F), and between HAdV-F (genotype 40 and 41) (n=32; 16.0± 9.172) and non HAdV-F genotypes (n=29; 13.97 ± 9.443) showed no significant difference (p = 0. 732; p=0.397).