Atul M. Walimbe

Human and swine hepatitis E viruses from Western India belong to different genotypes

BACKGROUND/AIMS Hepatitis E is endemic in India. Earlier, we showed prevalence of IgG antibodies to hepatitis E virus (IgG-anti-HEV) in different animal species and inability of at least one human hepatitis E virus (HEV) strain to infect pigs. In the US where hepatitis E is not endemic in humans, zoonotic spread of HEV was suspected as swine and human HEV were closely related and cross-species infection was documented. The present study attempts to identify and partially characterize swine HEV from India. METHODS Serum samples from 284 pigs were screened for the presence of HEV-RNA (nested polymerase chain reaction; PCR) and IgG-anti-HEV (enzyme-linked immunosorbent assay; ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. Two sero-negative pigs were inoculated with swine HEV-positive serum pool. RESULTS ELISA and PCR positivity were 42.9 and 4.6%, respectively. All Indian swine HEV sequences clustered with genotype IV. Pigs could be experimentally infected with swine HEV. CONCLUSIONS Swine HEV circulates in Indian pigs. In contrast to US and Taiwan wherein both human and swine HEV isolates belong to same genotype, Indian human HEV isolates belong to genotype I whereas genotype IV circulates in swine. Though experimental infection with Indian swine HEV was possible, at least one human HEV strain could not infect pigs.

Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993)

Seventeen Indian hepatitis E virus (HEV) isolates, representing epidemic and sporadic hepatitis E cases during 1976-1991, were sequenced in the RNA polymerase (RNAP) region. Five isolates were also sequenced in the non-structural hypervariable region of open reading frame 1. Open reading frames 2 and 3 were sequenced only for the prototype isolate. On the basis of the comparison of all the available sequences of the conserved RNAP region, the HEV isolates were divided into three genotypes, differing from each other by >15%. Genotype I included African and Asian isolates, whereas II and III were represented by Mexican and US isolates, respectively. Genotype I was further divided into four sub-genotypes. The majority of the Indian isolates (15/20), along with the Burmese and Nepali isolates, belonged to genotype IA. Genotype IB included HEV isolates from China, Pakistan and the former USSR and 2/20 Indian isolates, which represented the oldest (1976) HEV sequenced so far. Genotype IC included both the African isolates, whereas 3/20 Indian isolates formed genotype ID. Nucleotide sequence analysis of other regions of the HEV genome also placed isolates in the same genotypes. Both the Indian cities experiencing second HEV epidemics, after intervals of 8 and 10 years, showed shifts in the sub-genotypes found; from IB (Ahm-76) to IA (Ahm-84) and from IA (Kol-81) to ID (Kol-91). However, no major shift in the genotypes was noted. Overall, HEV genotypes appear to be segregated geographically.

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): selection pressure and molecular clock analyses.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Hepatitis B virus: prevalence of precore/core promoter mutants in different clinical categories of Indian patients

Summary. To determine the association of precore (Pre‐C)/basal core promoter (BCP) mutants with clinical outcome of hepatitis B in Western India, 192 hepatitis B virus (HBV) infected individuals were investigated. HBV‐DNA PCR positivity among asymptomatic hepatitis B surface antigen (HBsAg) positive carriers (61/100) was lower (P < 0.0001) than chronic hepatitis B (CHB), acute (P = 0.0001), and fulminant hepatitis B patients (P = 0.047). Pre‐C status was based on restriction fragment length polymorphism (RFLP, n = 153) and sequencing (n = 118). Prevalence of Pre‐C mutants was higher among carriers (23/61) than CHB (10/62, P = 0.0071) or acute (3/22; P = 0.037) patients. Children from carrier and CHB categories showed significantly higher circulation of Pre‐C‐wild than mutant HBV. Clinical manifestations were independent of BCP mutations (1762/64‐T/A). Hepatitis B e antigen (HBeAg) negative CHB patients [62.5% (15/24)] were circulating wild HBV. Higher HBV‐DNA levels were associated with chronic hepatitis and HBeAg positivity, whilst Pre‐C mutant positives had lower levels. BCP mutations did not affect HBV‐DNA levels. Multivariate regression analysis identified HBeAg (OR = 4.3) and Pre‐C mutants (OR = 3.1) to be associated with chronic hepatitis and carriers respectively. In a separate sub‐set analysis (n = 59), HBV‐DNA level was identified as the only variable. In conclusion, chronic or fulminant hepatitis B was not associated with Pre‐C or BCP mutants and switching over to Pre‐C mutant was beneficial for the infected individual in maintaining disease free status for extended periods.

Retrospective serological analysis of hepatitis E patients: a long-term follow-up study.

The aim of this study was to evaluate the persistence and protective role of antibodies to hepatitis E virus (anti‐HEV) after natural hepatitis E infection. A retrospective analysis of immunoglobulin G (IgG) anti‐HEV was performed in 37 patients followed‐up for 5 years after epidemics of HEV. Two patients with sporadic hepatitis E (HE) were followed‐up for 12 and 8 years. All patients infected during epidemics of HE were positive for IgG anti‐HEV at 5 years of follow‐up (geometric mean titre: 174.75). The two patients with sporadic HE were positive for IgG anti‐HEV at the end of 12 and 8 years of follow‐up (the IgG anti‐HEV titre was 1 : 200 in each patient). This study showed protection against disease by antibodies to HEV. It was therefore concluded that hepatitis E may be preventable by an efficacious vaccine.

Molecular characterization of three novel intergenotype norovirus GII recombinant strains from western India.

The phenomenon of recombination has been widely described among noroviruses (NoVs) in the past few years. In a NoV surveillance study conducted in western India, 3 novel and 3 known combinations of RNA-dependent RNA polymerase (RdRp) and capsid genes were identified in genogroup (G) II NoV strains. The present study pertains to the characterization of three novel intergenotype NoV GII recombinant strains. RT-PCRs were carried out for the amplification of nearly complete RdRp and complete capsid genes spanning ORF1/2 overlap of three strains followed by sequencing of the amplicons. The recombination event was confirmed by phylogenetic analysis using Bayesian MCMC approach, SimPlot analysis and Maximum chi(2) method. Three novel intergenotype (GII) recombinations of GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13 specificities were identified respectively in the strains PC03, PC24 and PC25 for the first time. The breakpoint in the novel recombinants was placed in the vicinity of the 20 bp ORF1/2 overlap, a common hotspot known to exist in NoV recombinants. The capsid genes of all of the 3 recombinants were closely related to their counter parts in reference strains however, a high degree of variation emerged in the polymerase genes especially of PC24 and PC25 in comparison to the reference strains.

Increased risk of hepatitis E in sewage workers from India.

Considering feco-oral transmission of hepatitis E virus (HEV), the risk of the infection was assessed among sewage workers. On the basis of the close contact with sewage, the participants (n = 147) were divided into sewage workers (n = 92) and others (n = 55); none used personal protective equipment (eg, coveralls, boots, gloves) Age-matched individuals from lower socioeconomic status and without any exposure to sewage were used as controls. IgG-anti-HEV positivity in enzyme-linked immunosorbent assay was significantly higher (P < 0.01) among staff members (83/147, 56.5%) than the controls (19%). A significant rise in anti-HEV positivity (P < 0.05) was recorded in sewage workers working for >5 years. Multivariate regression analysis identified contact with sewage as the independent variable associated with anti-HEV positivity. Strict adherence to good working practices must take top priority for protection of these workers from sewage pathogens.

Complete genome characterization of Genogroup II norovirus strains from India: Evidence of recombination in ORF2/3 overlap

Noroviruses (NoVs) are considered as important causative agents of non-bacterial acute gastroenteritis, worldwide. The data on NoV genomes, their diversity and evolution from Indian subcontinent are not available to date. The present study describes the characterization of full-length genomes of Indian NoV strains for the first time to establish their phylogenetic and evolutionary relationship with those circulating worldwide. Amplification of full-length genomes of three NoV strains (PC15, PC51 and PC52) was carried out using nine overlapping sets of forward and reverse primers. Full-length genomes of all of the three strains were characterized by phylogenetic, SimPlot, selection pressure and hydrophilicity analyses. The strain, PC15 was placed in the GII.4-Hunter subcluster. An intragenotype recombination event between ORFs 2 (new GII.4 variant) and 3 (Den Haag subcluster) of the strain, PC51 was detected for the first time in this study. The strain, PC52 showed the presence of commonly detected intergenotype recombination, GII.b/GII.3. A 16 amino-acid signature code (TDVVYYAGASQPRDDI) was identified in the ORF2 of recombinant GII.3 specificity strains, which may serve as a genetic marker for differentiation of these strains from non-recombinant GII.3 strains. The amino-acid substitutions in the ORF2 of PC51 and PC52 strains in comparison to the reference strains (Toyama1 and TV24) resulted in an increase in the hydrophilicity suggested alterations in the antigenic regions of Indian NoV strains. A unique pattern of amino-acid substitutions was observed within seven subclusters of GII.4 at 19 sites (including 13 sites under positive selection pressure) spanning entire ORF2. The study indicates adaptation of NoVs in the environment to escape the host immune response and to persist in the population. It also provides in-depth analyses of NoV genomes from India and determines the extent of conserved and variable features of the Indian NoV strains.

Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962–2005)

Dengue is a major health problem in India with all four serotypes represented. Recently there has been an increase in the occurrence of dengue-1 outbreaks. It is possible that there have been changes in the genetics of dengue virus-1 (DENV-1), either by fresh introductions or by evolution in situ. The studies on DENV-1 evolution so far have no Indian sequences included. To gain insight into the dynamics of DENV-1 in India, the envelope (E) gene of thirteen virus isolates representative of the period 1962-2005 were sequenced and analyzed together with the available sequences of 40 globally representative isolates. All the Indian DENV-1 isolates were found to belong to the American African (AMAF) genotype. With the addition of 13 Indian isolates, the AMAF genotype can now be called Cosmopolitan. The Indian isolates were distributed into four lineages, India I, II, III and the Africa lineage, now called Afro-India. Of these, India III was the oldest and extinct lineage; the Afro-India was a transient lineage while India I, imported from Singapore and India II, evolving in situ, were the circulating lineages. Despite the extinction and introduction of lineages, no specific codon site was observed to be under selection pressure. The rate of nucleotide substitution estimated for DENV-1 was 6.5 × 10(-4) substitutions/site/year, and the time to the most recent common ancestor (tMRCA) was estimated to be 78-180 years (1825-1925), similar to previous estimates. The tMRCA for the AMAF/Cosmopolitan genotype was 56-98 years (1907-1949), a period that covers World War I and II. The two imports from Africa (1953-1968) and Singapore (1964-1975) and an export to the Americas (1955-1965) prove that there have been changes in the lineage of the DENV-1 viruses circulating in India which has contributed to the global dynamics of DENV-1 evolution and perhaps to the changing epidemiology of dengue in India.

Full length genomes of genotype IIIA hepatitis A virus strains (1995-2008) from India and estimates of the evolutionary rates and ages.

With the changing epidemiology, outbreaks of Hepatitis A Virus (HAV) have been reported from different parts of India. To characterize HAV strains circulating in India (1995-2008), 6 full genome sequences of the predominant genotype, IIIA, were determined. Further, applying the Bayesian Markov Chain Monte Carlo (MCMC) framework to the full genomes of Indian HAV strains as well as other global strains (human as well as simian), we derived the mean nucleotide substitution rate and evolutionary timescales with emphasis on the age of genotype III and IIIA strains. The genomic length of all the 6 HAV isolates was 7464 nt excluding the poly A tract. Phylogenetic analysis confirmed that all the Indian isolates were close to Nor-21 (AJ299464) and HMH (AY644337) of subgenotype IIIA. The ORF of the isolates when compared within genotype III at amino acid level showed a highly conserved pattern. Under the best fit expansion population relaxed molecular clock model, the estimated mean substitution rate of the HAV full genomes (human and simian strains) was 1.73 x 10(-4) substitutions/site/year based on which the earliest transmission of HAV from simian to humans is estimated to have occurred about 3564 years ago. The mean substitution rate within human HAV full genomes under the same model was estimated to be 1.99 x 10(-4) substitutions/site/year. With this the mean age of genotype III strains was estimated to be 592 years while that of genotype IIIA was estimated to be 202 years. The time to the most common recent ancestor (tMRCA) of the Indian genotype IIIA isolates was calculated to be 116 years.