Preliminary Methods For Concentrating Hepatitis A Virus From Water Samples And Genetic Characteristics Of The Prevalent Strains Circulated In China

Hepatitis A, caused by Hepatitis A virus (HAV), is an infectious liver disease. There are six genotypes of HAV, but only a single serotype exists. HAV is mainly transmitted vial the fecal-oral route and hepatitis A outbreaks caused by contaminated food or water are serious health hazards to the mankind. Therefore, detection of HAV in food or water products is of importance to the food and water safety. However, it is difficult to directly detect the HAV due to its low density in water samples. Methods for concentration of HAV from water samples have become a hot topic in current researches.In this study, an electropositive Zetapor membrane followed by polyethylene glycol precipitation were used to concentrate the HAV from the viral spiked water, then the recovered HAV RNA was amplified by real-time quantitive reverse transcriptase PCR. Ultra pure water and mineral water spiked with Hepatitis A Attenuated Live Vaccine (HepA-L) were used as experimental samples, and non-inoculated water was used as negative process control. The HAV adsorption capacity on glass bottles and plastic bottles was evaluated.10%(weight/volume, w/v) polyethylene glycol (PEG)-1.0M NaCl generated a better recovery of HAV compared to other combinations. The electropositive Zetapor membrane was more efficient at the flow rate of 3L/h compared to other flow rate. The detection limit of the filtration method followed by the PEG precipitation was 300TCID50/1.5L(0.2 TCID50/mL). Mean recovery values for HAV using the optimal concentration method ranged from 0.38% to 0.88% depending on the initial viral loads. Lower quantity of HAV particles was absorbed to the glass bottles than the plastic bottles. And similar recovery was found between the ultra pure water and mineral water after PEG precipitation.In this study, the concentration conditions were optimized and the HAV RNA was detected using the real-time quantitive reverse transcriptase PCR. Both the ultra pure water and the mineral water seeded with HepA-L were used as the contaminated water. This study laid a foundation for the future research on the modification of the concentration methods for HAV in water samples.Only a single serotype of HAV exists so far, and the neutralizing epitope sites were mainly located within structural protein VP3 and VP1. Analyzing the genetic characteristics and the molecular evolution of the capsid protein VP3-VF1 region of HAV provides information on genotyping, the characteristics of the amino acid sequences, the origin time and evolutionary rate. Besides, this study can provide powerful theoretical and technical support for molecular evolution, control and prevention of HAV.In this study,39 HAV prevalent strains from 4 provinces of China and 2 HepA-L samples were collected. The nucleotide sequences of VP3-VP1-2A region of the samples was amplified by reverse transcription PCR after RNA extraction. The sequnces were further subjected to the homology and phylogenetic analyses, neutralizing epitope sites, selection pressure, evolutionary history and population dynamics.Phylogenetic analysis based on the VP3-VP1-2A region indicated that all the 39 detected HAV prevalent strains were clustered into subgenotype IA with 94.8%-100% nucleotide sequence identities and 99.3%-100%amino acid sequence identities, while the 2 tested HepA-L samples belonged to subgenotype IB with 90.6%-91.9% nucleotide sequence homology and 99.1%-99.4% amino acid sequence homology compared to the prevalent strains. No amino acid changes at published neutralizing epitope sites of VP3-VP1 region were observed, however, unique amino acid substitution were identified at the position of VP3-56(Serine→Proline) in 1 isolate, and VP1-112 (Threonine→Isoleucine) in 3 isolates, the latter one is closed to the neutralizing epitope site VP1-114. Negative selection was found in the detected sequences. Under the best fit model, the estimated mean substitution rate of HAV genotype Ⅰ in China was 5.56×104 substitutions/site/year, the time to the most common ancestor (tMRCA) of genotype I HAV isolates was calculated to be around 180 years ago. The Bayesian skyline plot showed the incidence of HAV went down gradually from the mid-1990s.The genetic characterization of the HAV isolates from China showed that in capsid regions the sequences were more conserved at amino acid level than that at nucleotide level. The isolated strains in this study were undergoing negative selection pressure. The evolution trends of genotype Ⅰ HAV in China were also exhibited in this research. This study laid a foundation for the molecular epidemiological research of HAV and the complete understanding of the HAV evolution.