Influence of Hepatitis E viral load and genotypes on pregnant urban dwellers of Bangladesh

Abstract

Background: Hepatitis E virus (HEV) often causing self-limiting hepatitis, appears to be a global health issue specially in Low- or Middle-Income (LMIC) countries. Pregnant women are more likely to experience severe morbidity and mortality, particularly, in South Asian countries including Bangladesh. Its pathogenetic basis is still not clearly understood. Objectives: The aim of the present study was to demonstrate the hepatitis E viral load, its genotypes, and IgG immune responses against the HEV virus, and to find out the association of these parameters with pregnancy related morbidity and mortality; for the purpose of determining HEV IgG antibodies utilizing dried blood spots (DBS) samples to design and validate a modified Enzyme linked Immunosorbent Assay (ELISA), to explore the molecular basis of binding affinities of HEV ORF2 wild type and mutant peptide with HLA alleles, and to determine the effects of mutation of capsid protein on disease severity using in-silico CD8 + T cell epitope prediction. Methods: One hundred and twenty-one women (pregnant, n = 57; non-pregnant, n = 64) with acute HEV infected hepatitis, attending tertiary care hospitals in Dhaka and Chattogram cities were enrolled in the study after collecting their respective sociodemographic and clinical data. Biochemical analysis e.g., serum bilirubin, alanine transaminase (ALT) and aspartate transaminase (AST) were carried out for all the samples. Seventy-eight HEV IgM-positive samples were subjected to reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) for estimation of viral copy number from their extracted RNA. Out of 62 real-time PCR positive samples, Sanger sequencing of HEV ORF2 was carried out successfully on 32 samples after purification of PCR products. IgG antibody for HEV was determined by ELISA and avidity indices were calculated. Dried Blood Spot (DBS) samples were prepared from HEV patients (n = 45), and compared with healthy controls (n = 20) in order to determine HEV IgG antibody by modified ELISA. Using the NetMHCpan 4.0 Server, epitopes of capsid protein encoded by HEV ORF2 were predicted for CD8 + T-cell. Both 9-mer peptide lengths and human leukocyte antigen (HLA) alleles were chosen and submitted. Molecular docking and molecular dynamics simulations were carried out for two common HLA alleles of Bangladesh. Results: In the late acute phase of hepatitis, there were significant negative correlations between log HEV copy number and log HEV IgG antibody index for both pregnant (r = -0.7971, p = 0.0002) and non-pregnant (r = -0.9117, p = 0.0002) women. When HEV-induced hepatitis reached its late acute phase, pregnant women revealed significantly greater serum log viral copy number and lower IgG antibody indices than non-pregnant women (p=0.0196 and p=0.0303, respectively). Additionally, compared to non-pregnant women, pregnant women with acute HEV hepatitis exhibited higher levels of cross-reactive IgG antibodies (p=0.0017). Four of the five HEV hepatitis patients that expired were pregnant. DBS and plasma samples demonstrated almost similar IgG antibody levels for HEV. At dilution 1:200, a highly significant correlation between plasma and DBS sample absorbances was found (R 2 =0.98; p <0.001), demonstrating real agreement between the two techniques. These procedures also showed linearity and showed no influence of haematocrit on test performance. The genotype for HEV was discovered as variant 1. A novel mutation, Q531L in 3 samples was identified. Significantly low levels of ALT (p= 0.001) for mutant HEV ORF2 531 Leu -containing peptide than that of wild type HEV (ORF2 531 Gln ) was observed. The 531 Leu containing epitope showed higher binding affinity than 531 Gln containing epitope for the HLA molecules. Conclusions: High viral load at the late acute phase of hepatitis can be assumed to be resulting from low level of HEV IgG that renders little neutralizing effect on HEV-infected pregnant women demonstrating higher morbidity/ mortality in genotype 1 HEV-infected pregnant women. A less- invasive collection strategy with a low-cost lab detection ELISA method has been developed for determination of HEV IgG by using DBS samples which is assumed to be much more promising nowadays. The binding free energy profile of mutant 531 Leu peptide with HLA alleles were considerably higher than that of wild 531 Gln peptide indicating the association of 531 Leu mutation with less morbidity and providing host protection. These results suggest that the Q531L mutation may be associated with decreased viral load and less severity of disease.