Identification Of Hepatitis B Virus Intergenotype Recombination From China

BackgroundHepatitis B is a disease of liver inflammatory lesions that may lead to multiple organ damage caused by the hepatitis B virus (HBV). Infection of hepatitis B virus is a serious challenge to global public health that the population of chronic HBV carriers is at the risk of potential adverse outcomes including developing hepatic decompensation, cirrhosis, and hepatocellular carcinoma.Hepatitis B virus is a partially double stranded DNA virus as a species of the genus orthohepadnavirus, which belongs to the hepadnaviridae family of virus. HBV is known as the smallest virus with the complete genome of3200bp length. By an intergroup divergence of more than8%in the complete genome, eight known HBV genotypes from A through H have been identified so far. In addition, two novel provisional genotypes are designed, one is the genotype I resulted from a complex pattern of recombination within genotype A、genotype C and genotype G; the other is the genotype J divergent from known human and ape genotypes. Moreover, based on an intergroup divergence of more than4%in the complete genome, different subgenotypes can be decided within a genotype. Associated with ethnicity and human migration, HBV genotypes have distinct geographical distribution. Genotype A is found in Europe, North America, South America and Africa, whereas genotype B and C is dominating in East Asia and Southeast Asia. Genotype D spreads worldwide including the Mediterranean, Middle East, South Asia and Oceania. Genotype F is more prevalent in South and Central America. The remaining three genotypes E, G and H are mainly found in Sub-Saharan Africa, the United States and Central America respectively. Recently, some virus strains similar to the strains defined as provisional genotype Ⅰ were also found in Laos, Vietnam, India and southwest of China.Recombination between different HBV genotypes is not rare, for instance the genotype Ba is generally considered as the recombinant between genotype B and C. With the rapid development of bioinformatics and recombination detecting software, the evidence of intergenotype recombination events have been identified successively in HBV phylogenetic analysis, such as C/D recombinants found in southwest of China and India, A/D recombinants circulating in Italy and South Africa, A/E recombinants from Cameroon and C/G recombinants identified in Thailand. However, the mechanism of HBV intergenotype recombination is still unclear. Recombination is considered as an important factor of genetic variation and a source of HBV evolution.ObjectiveIt is necessary to have a comprehensive screening for recombination in HBV genomes from China with high HBV infection rate. Thus, the aim of this paper is to identify the intergenotype recombination events and the rule of recombination from HBV genomes in China with the method of bioinformatics.Subjects and MethodsIdentification of HBV intergenotype recombination from China was carried on with the improvement of Fragment-typing. All complete genome sequences of human HBV from China including Hong Kong and Taiwan in GenBank were obtained except identical and artificial sequences at the website of National Center for Biotechnology Information (NCBI) in20th November,2013. In order to ensure the accuracy of computation, sequences with more than50unknown nucleotides or with the length of less than3000bp were eliminated from the dataset and the hypothetical EcoRI site was chosen as the starting of the genome. At length, up to1642human HBV complete nucleotide sequences were analyzed in this report.Then, consensus sequences of eight genotypes from A to H were generated using these complete sequences with reference genotype information learned from GenBank features and the related citations in PubMed. Eight consensus sequences were aligned by Muscle using MEGA version5.21. The multiple alignment of3257bp long was segmented into13subdivisions of250bp except for the last257bp subdivision. A database of104fragments collected from13subdivisions was formatted. The1642sequences as query sequences were done a local batch BLAST against the formatted database using BLAST package downloaded from NCBI. Fragment types, namely, genotypes of13corresponding fragments of each genome were defined on the basis of BLAST results. Sequences of the same13fragment genotypes were not considered as potential intergenotype recombinants and removed, leaving other sequences of different fragment types to be analyzed in following second screening for recombination. The remaining complete genome sequences were multiply alignment by Muscle conducting MEGA version5.21. The alignment was also spilt into13subdivisions equally of approximately260bp. A local BLAST of all fragments collected from these13subdivisions was done against another database of eight full length consensus sequences of genotypes from A to H. Consequently, genotype of each remaining sequence consisting of13fragments was described as13capital letters. As was mentioned above, sequences of13different letters were reckoned to be potential intergenotype recombinants. The mosaic fragments that genotypes were not in accordance with the majority of13fragment genotypes in each genome sequence were carried out for phylogenetic analysis using MEGA with the corresponding region of25reference sequences including8genotypes consensus sequences, two sequences at random each genotype and a HBV sequence from woolly monkey as an outgroup. Genotypes of the mosaic fragments were decided by the result of phylogenetic trees. Genotypes of13fragment of each remaining sequence were confirmed.Each sequence with putative recombinant was analyzed further to identify recombination breakpoints by applying SimPlot using similarity plotting, bootscanning and informative sites analysis with two consensus sequences of the parental genotypes and a fourth consensus sequence of another genotype as an outgroup. The recombined fragments were carried out for phylogenetic analysis with the corresponding region of25reference sequences including eight genotypes consensus sequences, two sequences at random each genotype and a HBV sequence from woolly monkey as an outgroup with a bootstrap value of75%.ResultsUp to1642human HBV complete nucleotide sequences from China including Hong Kong and Taiwan in GenBank were collected to be identified for genotype and intergenotype recombination by using fragment-typing. The number of genotype sequences from genotype A to D and genotype I was defined to be8(0.5%),634(38.6%),935(57.0%),30(1.8%),35(2.1%), respectively. In these genotypes, genotype B and C were dominant genotypes (95.6%) in China, and the sequences genotype B were all identified to be Ba subgenotype without Bj subgenotype.755recombinants were identified from1642HBV complete genome sequences. The results were described as follows:22B/C recombination types including676 recombinants,5C/D recombination types including75recombinants,3A/C recombination types including3recombinants,1C/I recombination type including1recombinant.31recombination types comprising755recombinants were identified of which four recombination types were identified to be novel types for the first time. And all recombination types in China involved genotype C.Recombination breakpoints were identified by SimPlot using similarity plotting, bootscanning and informative sites. Several studies have reported that the hotspot of recombination breakpoints is found to be located near the gene boundaries. In this report, we found that the distribution of recombination breakpoints seems to be consistent with regions reported in past studies.ConclusionIdentification of intergenotype recombination from human HBV complete genomes of China in GenBank was carried out for the first time. We employed fragment typing based on sequences similarity Blast and phylogenetic analysis, applied Simplot to identify the breakpoints of recombination. This method is suitable for the large scale analysis of recombination reducing the cost of time and computation.The geographical distribution of HBV genotype is obviously distinct because of ethnicity and human migration. The genotypes mainly circulating in China are A-D genotypes. Among these genotypes, genotype C is the most widespread genotype, genotype B is common in the south of China, while genotype D is restricted to northwest of China, especially in Xinjiang Province. Genotype A is a rare genotype in China. This is consistent with the distribution of genotype identified in this study.755complete genomes were identified as recombinants, with a proportion46%to the all1642genomes.31recombination types were described as following:B/C recombination type (71%,22/31), C/D recombination type (16.1%,5/31), C/A recombination type (9.7%,3/31), C/I recombination type (3.2%,1/31). It is reported that C genotype had a higher tendency for recombination among HBV genotypes and we found genotype C participated in all recombination types in this analysis. In consideration of genotype C that is widespread in China, co-infection between genotypes may increase the chance of recombination.634sequences belonging to genotype B were identified to be ten B/C recombination types and none of pure B genotype genome was discovered in this study. As might have been expected,624sequences were B/C recombinants of fragment type "BBBBBBBBCNBBB" that mainly circulated in East Asia and Southeast Asia with the recombination with genotype C. It is recommended that genotype B HBV should be divided into pure type Bj and recombinant type Ba in the form of intergenotypic recombination with genotype C in the genome part of precore and C gene. There were seven B/C recombination types involving second recombination that probably resulted from the recombination of Ba subgenotype and C genotype rather than the recombination of pure genotype B and genotype C as the absence of pure genotype B in China. A similar situation also appeared in C/I recombinant. Genotype I was generally considered as a new A/C/G recombinant genotype, and the C/I recombinant was the result of second recombination of genotype C and I instead of another A/C/G recombination with the pattern of different breakpoints.It showed that C/D recombination was another important recombination event in China. Learning from relevant sequence information and literature, C/D recombinants were mainly found in the northwest region of China, including Tibet, Xinjiang, Qinghai, Gansu and Sichuan. Tibet、Qinghai and west Sichuan as the part of Qinghai-Tibet plateau is the area inhabited by Tibetan nationality that represents an ethnic group same to India. As the settlement of Uygur and Hui minority, Xinjiang and Gansu were major spots on the Silk Road that linked China with the West in ancient times. Genotype D is widespread in India and Middle Asia. On different conditions, integration with C genotype that is common in Han nationality could lead to the different characteristics of the C/D recombinants.This study analyses all possible recombinants of complete HBV genomes from China available in GenBank and is of great value to further research of HBV genetic and recombination mechanism.