| Background:Enteroviruses (EVs) are members of the genus Enterovirus within the family Picornaviridae, order Picornavirales. The genus Enterovirus now can be classified to 12 species, Enterovirus A (EV-A) to EV-H, EV-J and Rhinovirus A to C. Most EV infections are asymptomatic or cause only mild symptoms such as Nonspecific rash or upper respiratory infection. However, some EVs can also cause a broad spectrum of other clinical illnesses, including acute flaccid paralysis, acute hemorrhagic conjunctivitis, encephalitis, aseptic meningitis and hand, foot, and mouth disease.EV-C including some of the EVs that can cause human diseases such as poliomyelitis caused by PV and acute hemorrhagic conjunctivitis caused by CV-A24v.In rencent yeas, some novel EV-C have been identified and caused several outbreak of diseases, which raised our concerns about other EV-C besides PV and CV-A24. EV-C96 is a novel type of enterovirus belonging to species C, its prototype strain BAN00-10488 was isolated form stool specimen of an AFP patient in Bangladesh. Several other EV-C96 strains were subsequently isolated from AFP patients or healthy individuals in Eurasia. Previous studies have shown multiple recombinations between EV-C96 and other EV-C in non-structural region. It has also been reported that different isolates of EV-C96 may have different cell tropism. Currently, there is no specific phylogenetic analysis about EV-C96 because of the limited number of strains, and we also barely know about the biological characteristics of EV-C96. In this study, we sequenced the complete genome sequences of EV-C96 isolated in China from 1997 to 2012, and studied the origin, evolution and biological features of EV-C96 on the molecular level and cellular level.Objective:By studying the EV-C96 isolated from China since 1997, we want to present the genomic characteristics of Chinese EV-C 96 as well as analyze the evolution informations about EV-96 such as the origin and evolutional rate to elucidate the evolutionary trend of EV-C96 on the genomic level. Further more, to discuss the biological characteristics of EV-C96 such as cell receptor, temperature sensibility as well as its relationship with human diseases. The results can provide basic information for the control and prevention of novel enterovirus-related diseases.Methods:To sequence the complete genome sequences of EV-C96 isolated in China by RNA extraction, primer designation, RT-PCR, then analyze the genome characteristics and evolutionary trend by bioinformatics methods such as similarity analysis and phylogenetic tree, analyzing the origin, evolution rate by using the Bayesian MCMC method. We also used molecular biology and cell biology method such as serum neutralization test, cell receptor inhibition test, temperature-sensitivity test and so on to study the biological characteristics of EV96.Resuls:1. The genetic distances of Chinese EV-C96 in VP1 region are among 0.093-0.226. All the Chinese EV-C96 can be divided as seven genotypes according to the cut-off value (>15% nucleotide diversity in VP1 region) of genotype. The EV-C96 strains from worldwide can be devided to cluster A, cluster B1 and cluster B2. The genetic distance between cluster A and cluster B1 are among 0.254-0.305, the genetic distance between cluster A and cluster B2 are among 0.235-0.297, the genetic distance between cluster B1 and cluster B2 are among 0.210-0.265.2. The evolution rate of Chinese EV-C96 is about 6.78×10-3/site/year, and the time for the most recent ancestor (tMRCA) could date back to 1972. The evolution rate of EV-C96 worldwide is about 5.31×10-3/site/year, the time for the most recent ancestor could date back to 1930. All EV-C96 strains worldwide fromed four lineages, lineage A-D, all Chinese strains belongs to lineage D.3. The total seroprevalence of some areas in Xinjiang is 65%, GMT is 1:82. The seroprevalence of kashi area is 70%, GMT is 1:82, the seroprevalence of hetian area is 60%, GMT is 1:196. There is statistical difference between the GMT of the two area by Mann-Whitney test (P<0.001).4. Strain 3-193 and strain GD809 can cause CPE only on RD cells, not on HEp-2 cells. Strain 1045,1072,1202F,882F and 3-156 can cause CPE only on HEp-2 cells, not on RD cells. T49 and 9-13 can cause CPE on both RD cells and HEp-2 cells.94H can not cause CPE on both RD cells and HEp-2 cells. When the HEp-2 cells were inhibited by anti-ICAM-1, the strain 882F cannot cause CPE on HEp-2 cells any more, so there is great possibility that EV-C96 can use ICAM-1 as a cell receptor on HEp-2 cells.5. Temperature-sensibility test shows that one of the Xinjiang EV-C96 strains 3-193/XJ/CHN/2011 is temperature sensitive, and all other Chinese EV-C96 strain are non-temperature sensitive. Thirteen specific amino acid site and nucleotide sites were found in the ORF of 3-193/XJ/CHN/2011, and three specific nucleotide sites were found in 5’UTR. |
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