Identificaiton Of The Intestinal Virome Of Children And Primates With Diarrhea Using Viral Metagenomics

Diarrhea is one of the most important public health problems, which is third infectious cause leading to death. Acute diarrhea is the popular and common disease in children population and often causes the death of infants, especially in developing countries, of which the major cause is the infection of microbes, including bacteria, viruses and parasites. With the wide utility of antibiotics and the improvement of water and sanitary, the diarrhea caused by bacteria and parasites are effectively controlled, the virus causing diarrhea, however, is still increasing, so as to attract more and more attention. The intestines of humans and animals host thousands of viruses, most of which are still unknown. Facing the infection of novel viruses, people have no suitable method to prevent and cure. Therefore, in order to conquer the infection of novel virus, people must discover the putative novel pathogenic viruses and do some researches for them, such as genome analysis, epidemiology, vaccine, and curing antibodies, which will be meaningful for the prevention, treatment of unknown viruses.Viral metagenomics is one the branch of metagenomics, which belongs to a new molecular biotechnology viral detecting method. This method can directly analyze all of the viral nucleotide in the clinical samples without needing to culturing the virus, using antibodies, or PCR amplification by specific primers. This method can not only identify the known viruses but also find the novel viruses effectively, so as to describe the composition and titers of viruses, and investigate the variation of virus population real-timely in a certain type of clinical sample, which will be important to the diagnosis of emerging and unknown pathogenic infectious diseases.Primates and humans are similar not only in physical features but also in social behavior. Nonhuman primates may carry many zoonotic pathogens. Therefore, this research used the viral metagenomics method to compare virus population in the feces from children and primate groups and excavate potential new diarrhea virus, which will be important to the diagnosis, treatment and research for viral diarrhea disease prevention and control of novel viruses outbreaks.The followings are the results of this study:1. The composition of intestinal virus population from children with diarrhea By constructing the viral gene library from diarrhea and healthy children and the Miseq high-throughput sequencing combining bioinformatics analysis, the intestinal virus population of children was identified. Results show that the intestinal virus population of children including: Caliciviridae, Adenoviridae, Picornaviridae, Reoviridae, phage, Parvoviridae, Anelloviridae, and other viruses. Virus community analysis indicated:(1) virus diversity from healthy controls is higher than that of diarrhea groups, while the virus titers in healthy controls are significantly lower than of diarrhea group. (2) all of the libraries from diarrhea group and healthy controls were positive for caliciviruses, and the virus titers in diarrhea group is significantly higher than those in healthy controls. (3) astrovirus is not main cause of diarrhea in this study.2. The composition of intestinal virus population in diarrhea primatesBy constructing the viral gene library from diarrhea and healthy primates and the Miseq high-throughput sequencing combining bioinformatics analysis, the intestinal virus population of primates was identified. Results show that the intestinal virus population of primates including: Parvoviridae, Virgaviridae, Adenoviridae, Picornaviridae, Phage, Picornaviridae, Geminiviridae, Circoviridae, Closteroviridae, Dicistroviridae, Iflaviridae, Astroviridae, Tombusviridae, Caliciviridae, and other viruses. Virus community analysis indicated:(1) There is no significant differences of the viruses composition and viruses titers between the diarrhea groups and healthy controls. (2) The viruses infecting mammals in the primate libraries are mainly the parvoviridae and anelloviridae, however, the virus titers of the two groups have no significant difference (3) The recent discovered picobirnaviridae virus is widespread in the gut of primates. (4) Astrovirus is not the main cause of diarrhea of primates in the study (5)The plant viruses in the primates gut have property of variety and higher titers.3.Comparing the virus population from children and primatesResults indicates:(1)The virus composition from primates is more diverse than that from children. The virome of primates include more than 15 virus families, while the virome of children only include 9 virus families.(2)the plant viruses are the main part of primates’virome, while the mammalian viruses account for the main part of children’s virome, which may because of the difference of diet of children and primates.(3)Generally, the virus titers in primates gut is higher than that from children.4. Identification of novel viruses from children and primate virus populations (1) AstrovirusesTwo different novel astroviruses (HAstV-JS1 and HAstV-JS2) and one novel astrovirus (MAstV-JS) are discovered from children and primates, respectively. Phylogenetic analysis shows that there is no closely related relatives of these three astroviruses in GenBank, which suggests that these three astroviruses belong to novel viruses. PCR detecting indicates that the positive rate of HAstV-JS1 in diarrhea children is 12.2%, while the positive rate of HAstV-JS2 in diarrhea children is 10%. The positive rate of MAstV-JS in primates group is 3.5%. All of the 3 astroviruses in healthy groups are negative.(2) NorovirusesBy sequence analysis, there are several types of noroviruses in the virome of children and primates. De novo sequence assembly produces 4 noroviruses with full or nearly full genomes(NoV-JS1, NoV-JS2, NoV-JS3 and NoV-JS4).These noroviruses share more than 95% sequence similarity with noroviuses from GenBank. Phylogenetic analysis shows NoV-JS1 and NoV-JS2 clustered in the same group and are closely related with the norovirus strain (KC894943) which is isolated from Guangzhou, China. Nov-JS4 are the relative of the norovirus (KF306214) isolated from Liuzhou, China. NoV-JS3 clusters with another Guangzhou strain (KF989075).Geneious software assembly finds a norovirus from primate with full genome, sequence analysis shows that this primates norovirus share 85% sequence similarity with the only primate calicivirus from GenBank.(3) Picornaviridae and PicobirnaviridaeOur results indicate that picornaviruses are associated with children diarrhea. However, picornaviruses are not associated with primate diarrhea. No virus from picobirnaviridae are discovered, which suggests that picobirnaviruses are not related with the children diarrhea in the present study. While the picobirnaviruses are associated with the primate diarrhea.(4) Novel HEV-like virusIn the current study, an HEV-like virus (MHEV-JS) with full genome of 7289bp is isolated from the fecal samples of primates. The genome contains three open reading frames, which is similar to the other human HEV, while sequence similarity analysis based on the capsid protein sequence shows that MHEV-JS only has about 30% sequence similarity with the other HEV strains. Phylogenetic analysis and base composition analysis both indicate MHEV-JS belongs to virus that can infect vertebrate, suggesting MHEV-JS does not originate from the food primates eat. PCR detection using the primers designed based on the sequence of MHEV-JS indicates MHEV-JS are positive in 5 diarrhea fecal samples and 2 healthy control fecal samples, all which are from the same sampling site, suggesting MHEV-JS has no significant association with diarrhea of primates.(5) Circovirus and Circo-like virusesIn the present study, one circovirus (CycV-JS) and one circo-like virus (GemyV-JS) are discovered from children virome and one circo-like virus(MGemyV-JS) is found in primate fecal sample. GemyV-JS and MGemyV-JS belong to Gemycircularivirus, while CycV-JS belongs to cyclovirus. The three viruses all code one Rep protein and one Cap protein. Phylogenetic analysis GemyV-JS clusters with dragonfly gemycircularvirus, sharing 49.4% sequence similarity with it based on the Rep protein sequence. MGemyV-JS clusters with a strain of germycirularvirus isolated from human plasma, sharing 48.5% sequence similarity with it based on the Rep protein sequence.CycV-JS clusters with cycloviruses and shares 56.3%-58.9% sequence similarity with the other cycloviruses over the Rep amino acid sequence. PCR detecting using the primers designed based on the three viruses indicates the three viruses has only one positive sample, respectively, suggesting these viruses are not related with diarrhea.(6) A novel adenovirus from primateA novel primate adenovirus (SAdV-Ch1) is isolated from primate fecal samples, of which the complete genome is determined. The genome size, genomic structure, and base composition of SAdV-Ch1 are similar to human group A adenovirus (HAdV-A), but different from the other simian adenoviruses from primates from GenBank. Phylogenetic analysis over the complete genome and 5 main genes indicates that SAdV-Ch1 clustered closely with four adenovirus strains from HAdV-A and genetically far from the other simian adenovirus strains. Recombination analysis reveals that SAdV-Ch1 is involved in a recombination event where recombination occurred between SAdV-ch1 and HAdV-61 that generate the recombinant HAdD-31. Our results also suggests that SAdV-ch1 has the potential of cross-species infection.Conclusion:(1)Using viral metagenomics method, the virus population of children and primates with diarrhea is successfully illucidated. (2)The diversity of virus in the virome of the primates is higher than that from children.(3)Three novel astroviruses, one novel HEV-like virus, three novel circo-like viruses, one novel adenovirus are identified from the virome of children and primates.(4)The novel adenovirus from primates has potential of cross-species infection.