The Influencing Factors Of Nervous System Infection Caused By Enterovirus 71-A Preliminary Analysis

1. Background and ObjectiveHuman enterovirus 71 (EV71) belongs to the enterovirus genus A within the family of picornaviridae. It is the main etiological agent of hand, foot and mouth disease (HFMD) in infants. EV71 infection usually causes mild hand, foot and mouth disease or herpangina. However, more and more reports reveal that EV71 infection can cause severe neurological syndromes, including aseptic encephalitis, meningoencephalitis, brainstem encephalitis, poliomyelitislike acute flaccid paralysis. severe neurogenic pulmonary edema and even death. EV71 was first described in 1969, after its isolation from a two-month-old infant with aseptic meningitis in California, USA, several EV71 epidemics with high mortality rates occurred in different regions, for example in Bulgaria, Malaysia, Singapore, Japan and Vietnam. According to the early warning information announced by Health Planning Commission of Guangdong Province in May 17 that there have reported a total of 88,366 cases of HFMD up to May 16, among which three cases were died. Compared with the same period in 2015 (51,327 cases), the number of cases increased by 72%, EV71 virus caused the highest death rate. As one of the main pathogens of HFMD, EV71 has become a worldwide public health threat. It is the most serious central nervous system toxicity pathogen instead of poliomyelitis virus. Therefore, it is very urgent to explore the susceptible factors of severe EV71 infection from epidemiological perspective, analyse the mutation of epidemic strains, so as to provide some strategy for the prevention of EV71.From the perspective of molecular biology point of view, EV71 is an RNA virus, the lack of proofreading exonuclease function in the replication process may leads to a higher error rate, almost each copy of a gene have a spontaneous mutation..Due to VPl’s high diversity and less involved in the reorganization and is considered to be enterovirus evolution of the most significant gene region. Studies have shown that there is a correlation between VP1 variations and nervous system infection caused by EV71. There may be a mutation of a single point who decided the viral neurotoxicity, or may be more than one point mutation result in it. A study found that there is an important amino acid mutations Ala 170 Val on VP1, believed this mutation makes the virus protein structure changes, altered virus receptor binding capacity. Therefore, we aim to verify whether EV71 VP1 variation was related with nervous system infection through a larger sample of people screened.In addition, a study about the TLR-5 and TLR-6 receptor expression brain tissue with nervous system damage caused by EV71 infection found that high expression of viral receptor in brain tissue may leads to nervous system damage. Another study have confirmed there is a correlation between EV71 receptor and nervous system damage caused by EV71. The first step of virus entry into cells is bingding with the cell surface receptors, The main way of virus enters the cell is the virus receptor-mediated endocytosis or RNA release, so we guess that patient’s own receptor quantity, the interactions between viral proteins receptor and virus itself may also affect the nervous system infection by EV71.P-selectin glycoprotein ligand-1 (PSGL-1), Human scavenger receptor class B2 (SCARB2), Vimentin (VIM) were currently known as EV71 receptors. In recent years, more and more studies have reported that VIM plays a vital role in pathogen adsorption, endocytosis, or in the migration process. VIM is a newly discovered EV71 receptor, which is plenty of expression in human brain microvascular cells, it is necessary for mediating E.coli bacterial invade the host cell. EV71 infection is similar to this invasion. So we guess EV71 is mediated by VIM for entering to the human brain microvascular endothelial cells, followed by infected the nervous system. Because the composition of EV71 infection in patients with nervous system infection is in a relatively stable trend over these years, according to reports in the existing literature,we concluded that the virus variation and the patient’s own receptor differences are factors which influencing the EV71 infection of the nervous system, they may have a the independent effect respectly or interaction effect.This article mailnly studied the epidemiological analysis of EV71 in Guangdong province from 2008 to 2010, and clarified the role of VIM in EV71 infection of HBMEC. It is useful to explain the fators influencing EV71 infection of nervous system, and provide some clues in the prevention and control of EV71 or nervous system damage caused by EV71. This study is not only helpful to analyze the genetic evolution and molecular epidemiology characteristics of EV71; it also useful to establish early warning signal for the nervous system of EV71 infection meaningful, as well as of early detection of the susceptible ones, so as to take effective preventive measures timely to reduce the incidence of nervous system infection has important significance.2. Methods(1) The epidemiological analysis of EV71 among 2008-2010 in Guangdong provinceCollecting samples sent to Guangdong Province Disease Prevention and Control Center tested (including feces, throat swab, cerebrospinal fluid, etc.) among 2008-2010 from many urbans in Guangdong, a total of 194 cases using quantitative PCR and virus isolation techniques diagnosed with EV71 after the infection, according to the difference of symptoms,we devided them into non-nervous system and nervous system groups. RNA was extracted from collected samples or cultured virus, then reverse transcripted it into cDNA, we amplified full-length EV71-VP1 using polymerase chain reaction technology, then conducted sequence alignment and established phylogenetic tree with MEGA software (version 5.0) to confirm the genotype of EV71. The association between severity of clinical symptoms and sex, age, viral genotype and VP1 variation was also analyzed using logistic regression. Use DNAman to find the variation of VP1, we difined a site of more than two amino acids was variant.(2) The establishment of human brain microvascular endothelial cells VIM knockout cell linesFrom US National Center for Biotechnology Information (NCBI) database, we firstly found the gene sequence of VIM and its CDS region. Then we analyse VIM gene structure, and make clear of CDS exon portion. In accordance with the nature of the gene itself, selecting a candidate to be knockout site after identifying the target site was designed and synthesized a pair of DNA Oligos. The sgRNA connected to LentiCRISPRv2 plasmid vector and construct Cas9 expression vector. The constructed plasmid object package into lentivirus, and then infect HBMEC. Finally we select the VIM-KO cell lines who stably express.(3) The effect of VIM in EV71 infection of nervous systemAfter amplified and titrated EV71 in RD cells, HBMEC normal and VIM-KO cell were infected with the virus. The cell pathological effects (CPE) was observed every day. Using the fluorescence quantitative PCR to detect the difference of EV71 virus adsorption capacity, replication, and release levels in normal and VIM-KO cells. The VP1 protein synthesis differences between HBMEC normal cells and VIM-KO were also detected.3. Result(1) The epidemiological analysis of EV71 among 2008-2010 in Guangdong provinceThe genotype of the predominant epidemical strain was C4a in Guangdong from 2008 to 2010. However, this subtype had already differentiated into 4 subgroups (C4a1-C4a4).(2) The time and region distribution of the EV71 strainsFrom the time distribution of the various strains showed:in 2008 four genotype were exsited, but mainly concentrated in C4a1; in 2009 were C4a1 and C4a2; and in 2010, excluded a strain from Zhanjiang belonging C4a4, the others were C4a1. From a geographical aspect:in most areas the genotype were C4a2 C4al type; only in 2008, Dongguan ramets 009-08-M alone constitute C4a3; C4a4 also appeared at only Foshan and Zhanjiang. In general, the distribution of these virus strain did not have a region and time trends.(3) EV71 VP1 A289T variation was related with the nervous systemWith increasing age, the incidence of severe cases was significantly decreased (P <0.05),91% of critically ill patients were less than 4 years old. We further analyse gender, age, virus type, the severity of the impact of VP1 variation of clinical symptoms by Logistic regression. The results showed that here was no correlation between clinical syndrome and sex or viral genotype; age and virus A289T mutation and clinical manifestations are closely related. the severity of symptoms was negatively correlated with age.Variation of 289A can easily lead to severe cases, increasing the risk of infection (P<0.05, OR=2.328,95% CI 1.163-4.659).(4) The successfully establishment of human brain microvascular endothelial cells VIM knockout cell linesUsing CRISPR-Cas9 and lentiviral vector technology to bulid a knockout model, then packaging lentivirus of VIM-KO and control. After the lentivirus infected HBMEC for 48 hours, we observated that the expression of GFP was nearly 70% to 80%. Screening a stable HBMEC VIM-KO and control cell lines with puromycin. In order to verify the effect of knockout, the specific antibodies targeted VIM were used to detected the expression of VIM in HBMEC VIM-KO and normal cell. It was found that the expression of VIM in VIM-KO decreased more than 90%, indicating that the VIM knockout achieve a desirable result.(5) VIM plays an important role in EV71 infection of nervous systemAfter the EV71 enters the cell, as it cause damage to cells, it will lead to changes in cell morphology. After infected the control HBMEC for 24h, the cells appeared shrunken, rounded or broken; with prolonged duration of infection((48h), some cells already floated in the culture; by contrast, with a same MOI of EV71 infecting VIM-KO, after 24h the cell morphology is merely changed, even to the 48th, the cell morphology changes very little, only a small portion of the cell begins to circle. It showed that EV71 infection can cause lesions effects to the normal HBMEC, but VIM knockout cells not as severe as the normals. The first step of EV71 invade to cells is adsorbed on the cell surface. Comparing and detecting differences between HBMEC and VIM-KO of the amount of viral nucleic acid which surface fluorescence quantitative PCR, the results showed that compared with the normal ones, after VIM knockout EV71 cell surface adsorption decreased by 40%. EV71 infected cells after 24h and 48h, the amount of virus in normal HBMEC year increased 3-fold and 8-fold, respectively, and VIM-KO cells replicate very little,24h after only 1.3 times of that in the Oh, after 48h, viral load is only Oh twice about. After EV71 infection HBMEC 48h, VIM knockout cells EV71 virus nucleic acid amount control cells 1/12. Cell culture supernatant EV71 virus nucleic acid test results show that the amount of cell knockout VIM 1.4 times less than the control cells. EV71 virus nucleic acid in the cell replication and protein synthesis are substantially synchronous, the difference in the cell by detecting EV71 in HBMEC control and knockout protein synthesis, our result showed that the expression of VP1 protein in VIM-KO is also great less than the HBMEC normal cell.4. Conclusion1. During 2008 to 2010, the genotype of EV71 predominant epidemical strain was C4a in Guangdong province. Futhermore, this subtype had already differentiated into 4 subgroups (C4al-C4a4). the distribution of these virus strain did not have a region and time trends.2. EV71 VP1A289T (alanine 289 threonine) variation were related with the infection of nervous system. Virus 289A easily induced the nervous system infection caused by EV71. The A289T mutation sites obtained from 194 cases of EV71 infection about the molecular epidemiology in Guangdong Province. The site was f discovered who is closely related to nervous system infection. Further study of this site have a practical significance for prevention and control of EV71 infection in Guangdong Province or in China.3. VIM is demonstrated as a surface receptors EV71 in HBMEC, by binding to the receptor, EV71 could invase the HBMEC, and caused cytopathic effect; affected the adsorption capacity in the cells This paper proposed the hypothesis path of EV71 infection of the nervous system:EV71 cross the blood-brain barrier by VIM receptor-mediated RNA release into HBMEC cells, and ultimately infection of the nervous system.The study will be helpful for revealing the mechanisms of the nervous system infection caused by EV71.4.This study combined study the variation of the virus and the viral receptor. We conclude that the variation of the virus itself and receptor VIM work together to affect the EV71 infection of nervous system. It provides a new idea for the study of mechanisms of EV71 infection of the nervous system.