Molecular Epidemiology and Genetic Characterization of Measles Virus in Pakistan

Abstract

Measles is highly infectious and fatal viral disease still responsible for more than 150,000 annual mortalities among children in the developing countries. In Pakistan, measles is endemic for years and comprehensive data about the disease burden and the viral genotypes is not available. The objectives of this study were to investigate the molecular epidemiology and genetic diversity of measles virus causing infections in Pakistani population. A total of 15,311 samples including 15,081 serum samples, 32 oral and 198 throat swabs were collected from suspected cases during 1st January 2013 to December 2015 reported across all provinces of Pakistan. The highest disease burden was noted in 2013 due to wide-spread outbreak in the country represented by 10,890 samples. The Sindh province was severely affected with 45% of total reported cases followed by Punjab and Khyber Pakhtunkhwa (KP) with 38.15% and 7.23% cases respectively. The outbreak was contained in Sindh reflected by a significant decline in the number of cases (n=1,572) notified during 2014 and 2015 except Karachi where cases continued to appear over three years. In Punjab six (Bahawalpur, Gujranwala, Lahore, Okara, Rawalpindi and Sialkot) and in Baluchistan four districts (Lasbella, Naseerabad, Quetta and Zhob) remained infected throughout the study period. In contrast, Peshwar was the most heavily infected district in Khyber Pakhtunkhwa province with sporadic cases notified from other districts. In 2013, most of samples were received during January and February compared to 2014 and 2015 where a slight increase was reported during April to June compared to the first quarter of the year. The coverage of immunization remained suboptimal indicating a single vaccine dose received by 51% children whereas only 21.9% children received two recommended doses of vaccine. The male patients remained dominant compared to females, with male to female ratio of 1.32:1, 1.13:1 and 1.16:1 during 2013, 2014 and 2015 respectively. The laboratory testing revealed that the overall IgM positivity rate remained 64% and IgM antibodies were detected in 52%, 9.3% and 2.5% patients reported during 20132015, respectively. Out of total 15,081 samples screened for IgM antibodies, 36% male and 28% females were found positive. The rate of positivity declined with increasing age and the children below 60 months of age represented the most infected age group. A total of 230 samples, including 198 throat and 32 oral swabs, were processed for detection and genetic characterization of measles virus. Thirty two (16.16%) throat swabs that were negative for measles virus RNA on real-time PCR were cultured on Vero/SLAM cells, out of which 22 (68.75%) samples yielded typical cytopathic effect of measles virus i.e. syncytia formation within 4-6 days of inoculation. For phylogenetic analysis and genetic characterization, 208 samples (166 throat, 20 oral swabs and 22 culture isolates) were processed. The complete H-gene of 63 isolates recovered from 50 throat swabs and 22 culture isolates and partial fragment of N-gene in 171 samples including 132 throat swabs, 17 oral swabs and 22 culture isolates was successfully amplified. Sequence analysis of H-gene revealed that the PAK sequences shared 99% - 99.46% nucleotide and 99.4% - 99.1% amino acid homology with the WHO reference strains, whereas the nucleotide and amino acid homology within the study strains was found as 99.8% and 99.1% respectively. A total of 21 amino acid substitutions were found in all PAK measles virus strains identified in this study. Out of these, five substitutions were found in antigenic sites at amino acid position 178, 307, 309, 400, and the fifth one was found at amino acid position 397. Overall, the mutation rate for synonymous and non-synonymous mutations remained 0.4% and 0.1% respectively. The partial sequencing of N-gene performed on 171 samples revealed that 168 viruses belong to genotype-B3 and 3 were classified as genotype-D4. The B3 strains showed an overall heterogeneity of >1.8% at nucleotide level and 2.3% - 3.5% nucleotide divergence from WHO reference strains. On the basis of phylogenetic analysis, all B3 viruses were divided in to 2 genetic clades. Clade-1 contained seven viruses isolated during 2014-15 in Baluchistan and had 0.9% nucleotide divergence from the members of clade 2. All of the viruses isolated from other provinces during 2013-2015 grouped into clade-2 which was further subdivided into two groups. Group-1 was further subcategorized into four lineages. The nucleotide divergence between lineage 1, 11, 111, and IV was 0.7% - 0.3% whereas viruses belong to group 2 clustered with New South Wales and Stockholm strains. This is the first report on the genetic characterization and diversity of measles virus genotypes prevailing in Pakistan. The data submitted in this thesis will not only be helpful to monitor the possible changing endemic measles strains but also to map the transmission pathways to elucidate the extent of outbreak potential in the less endemic regions in future. Furthermore, it will also help to advocate implementing a robust surveillance program for measles in Pakistan including large-scale sero-survey to determine the anti-measles immunity profile in the most vulnerable population of children less than five years of age.