East China Gene Type �� Hepatitis E Virus Zoonotic And Evolutionary Origins Of Research

Hepatitis E virus (HEV) is a pathogen of fecal-oral transmission which accounts for acute hepatitis E (HE) in the general population. HEV can lead to large-scale epidemics due to fecal contamination of drinking water in developing countries with poor sanitation as well as smaller outbreaks and sporadic cases over the world. Sporadic cases induced by zoonotic genotype-III and genotype-IV HEV are increasingly popular, which are maybe attributable to food-borne transmission.China is the area highly endemic for HEV. The largest outbreak of HEV worldwide occurred in Xinjiang Uygur Autonomous Region (XUAR) between 1986 and 1988. According to the bulletins on national epidemic situation issued by Ministry of Health of People's Republic of China, HE is the only increasing one among legally-reported enterically-transmitted diseases domestically from 2000 to 2008. In China Mainland, genotype-IV HEV has been dominant and swine is proved to be a principle reservoir. The epidemic situation of genotype-IV HEV in human population could be possibly affected by zoonotic transmission with swine HEV, which is widespread in numerous herds and evolves rapidly. The present study is designed to explore the evolutionary and demographic history of genotype-IV HEV using the combination of epidemiology and bioinformatics. It would contribute to forecasting of HE epidemic and establishment of prophylaxis towards HEV. The study could also be used for reference to the evolutionary analysis of other pathogens.Based on the clinical epidemiological investigation of HE cases and cross-section study within swine herds, the present study was conducted in Eastern China, including Shanghai, Deqing (Zhejiang Province), Anqing (Anhui Province) and Haimen (Jiangsu Province). Initially, the distribution of time, region, and population within human HE cases and HEV-infected pigs was evaluated and the temporal identity of HEV prevalences between them was determined. Then human HEV and swine HEV were isolated for phylogenetic reconstruction. Finally, the nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using relaxed molecular clock theory and Bayesian inference based on Markov chain Monte Carlo (MCMC). The host population (human and swine) dynamics was inferred using best-fit demographic model.Sporadic HE cases in Eastern China are mainly induced by genotype-IV HEV. There were more male patients than female ones (2.82:1). The majority (84%) of the cases ranged from 30 to 70 years old, in which 1/3 was 50-60 years old. The temporal distribution was demonstrated by the concentration of cases (63%) between Jan and Apr. The positive rate of HEV RNA in the first-sampled serum (sampled within 0-45 days after the onset, median was 9 days) was 55%-64%. But it declined rapidly to 28%-38% in the second-sampled one (sampled within median time of 19.5 days after the onset) and 4%-8% in third-sampled one (sampled within median time of 24.5 days after the onset), respectively. Employing Kaplan-Meier survival analysis, median detection time was estimated as 21 days and 29 days after the onset based on the temporal prevalence of HEV RNA in singly-sampled serum and serially-sampled ones respectively. It meant that the viremia in 50% of HE patients could remain detectable up to 21-29 days after the onset. It is implied that HE patient could still act as reservoir even after the duration of biochemical hepatitis.Genotype-IV HEV is also widely prevalent in swine herds in Eastern China. HEV RNA could be detected throughout the productive life of the pigs but the prevalence varied with time of life. It was the highest in the swine herds of 4-6 months, amounting to 33%-42% in feces and 4.8%-5.6% in biles respectively. Significant difference of HEV detection was observed in geographic distribution. The detection rates of Shanghai, Anqing and Haimen which all locate along the Yangtze River, were higher than that of Deqing which is far from the Yangtze River. Seasonal pattern was also demonstrated by the highest prevalence of 6%-8% between Sep and Oct and lowest one of 3.7%-5% between May and Jun. It could be interpreted as the seasonal pattern of swine HEV transmission. Interestingly, there was great similarity between the dynamics of swine HEV detection and human HE cases, but temporal distribution of the former is ahead of that of the latter by 3 months. Cross-species transmission of HEV was implied.In the present study, the HEV strains isolated in Eastern China were all grouped into genotype-IV and of 84%-85% identities with each other. The genetic diversity among human HEV isolates with great probability of C←→T transition was higher than that among swine HEV isolates with closer probabilities of transition and transversion. Regardless of isolation from different regions, human and swine genotype-IV HEV shared high similarity. All the isolates were clustered into 9 groups with identities of 94%-98% and 81%-90% within and between the groups. Each cluster was consisted of human HEV as well as swine HEV. It was noted that some human and swine isolates were clustered with bootstrap values of almost 100% and posterior probability of 1, in which cross-species transmission was demonstrated.Based on the general GTR model of nucleotide substitution and a relaxed molecular clock, Bayesian Skyline Plot (BSP) gave the evolutionary rate of 1.63×10^-3 substitutions/site/year and 6.94×10^-3 substitutions/site/year for human HEV and swine HEV respectively, which corresponded to estimated date for tMRCA of 1820s and 1970s. It was the first time to determine that genotype-IV HEV had been spreading since 1820s in Eastern China, which was consistent with the opening of local ports for trade with the outside world in Qing Dynasty. Exotic importation of HEV was implied. The tMRCAs of human HEV in the regions locating along the Yangtze River are earlier than that in the region far from the Yangtze River. But the tMRCAs of swine HEV in different regions were similar and much later than the tMRCA of human HEV. It suggested that original reservoir in Eastern China was maybe human beings. After a long time of circulation in human population the virus invaded pig herds, cross-species and bilateral transmission occurred consequently. The present study also estimated and reconstructed the demographics of genotype-IV HEV. The effective number of human population infected with genotype-IV HEV in Eastern China increased remarkably since 1990s but that of swine population infected decreased in recent years. Attributable to both of the fast growth rates of genotype-IV HEV isolated from human and swine and the risk of cross-species transmission, the epidemic of HEV locally will still remain considerable.