| Numerous novel viruses have been found in the development and application of metagenomics technology.Nairovirus-like is a new genus in Bunyaviridae.In 2008,American researchers discovered the first virus-South bay virus(SBV)in Ixodes scapulars by metagenomic technology.At present,only outh bay virus,Norway nairovirus 1(NNV-1),Grotenhout virus(GTHV)and Pustyn virus(PTV)have been found.Importantly,there are no virus reports infecting humans of this family worldwide.In this study,we found the sequence of Beiji nairovirus(BJNV)in Nairovirus-like genus from the whole blood of tick-biten patients using metagenomic data.The whole geomes were obtained by PCR method.Secondly,we isolated and identified the virus;thirdly,we established SYBR fluorescent-quantitative PCR method with high sensitivity,strong specificity and good repeatability to detect the blood samples of tick-biten patients.The demographic data of positive samples were analyzed retrospectively to summarize the clinical characteristics and other data,so as to provide basic data for the clinical diagnosis and public health safety to better assess this disease.Then,IgG and IgM of the patients infected with BJNV alone were screened to observe the change of antibody titer successfully expressing the purified BJNV-N protein.Finally,we evaluated the epidemic distribution of the BJNV in different animals through established method,which provided scientific data support for the research on the epidemic situation and host distribution of BJNV.1.In this study,BJNV infecting human,was found in the blood of tick-bitten patients using using metagenomic sequencing method.The whole genome of the virus was amplified and assembled by RACE and nested-PCR.It was proved that the whole genome of BJNV was composed of S segment(3715bp)and L segment(14851bp).BJNV was isolated and from human hepatoma SMMC-7721 cells and identified by electron microscopy.The virus was round shape,with a diameter of 80-110nm and an envelope,which was identical with Bunyavirales.2.The amino acid and nucleotide sequences of BJNV were analyzed by systematic bioinformatics technology,and the genetic evolution status of BJNV was determined.The homology analysis of amino acid and nucleotide sequences showed that BJNV shared nearly 100%homology with BJNV HLJ2015,suggesting this strain virus was belonge to the same virus.Bioinformatics confirmed that the 5'-UTR and 3'-UTR ends of the L and S segments have the same reverse terminal complementary conserved sequences(5'-UCUCGAAGA ande 3'-UCUCAGAGA).This structure is highly conserved with the 5'-UTR and 3'-UTR ends of Nairovirus-like,and is also highly conserved with the CCHFV ends of Nairoviridae Family.There are similar motifs,ovarian tumor domain,zinc finger motif,leucine zipper motif,N-terminal and C-terminal conserved regions in the L segment of BJNB genome compared with the sequence of Nairovirus-like.Phylogenetic analysis of S-amino acid and L-amino acid showed that the virus belonged to Nairovirus-like Family(Bunyavirales).3.A SYBR fluorescent-quantitative PCR method with high specificity,sensitivity and reproducibility for BJNV was established.The detection range of this method is 101 copy number/ml),the slope is-3.13,the constant is 34.87,R2=0.999.Copy number formula:10(34.87-y)/3.13(copy number/?L).SYBR-q PCR method and cell immunoflurescence results showed that BJNV could proliferate in wish,Vero,BHK-21and SMMC-7721 cell lines.4.In this study,129 BJNV positive patients from 658 tick bite patients was selectede using SYBR-q PCR method,including 67 infection patients alone.A total of67 infection patients alone had a definite history of tick bites(100%)mostly male(70.1%),with an incubation period of 3 to 7 days.,and the career all were field workers(100%),who were bitten from May to July(65.7%),with aged from 40 to 60(77.6%).These patients was mostly male(70.1%),with an incubation period of 3 to 7 days.The main clinical symptoms were fever(100%),headache(99%),mental malaise(62.7%),coma(62.7%)and fatigue(53.5%),a few patients had muscle pain(44.8%),loss of appetite(35.8%).The blood routine,coagulation function and biochemical indexes of patients were within the normal range.However,74.4%of patients with BJNV infection alone showed elevated C-reactive protein,69.2%of patients showed decreased albumin,42.4%of patients showed elevated glucose,38.5%of patients showed elevated serum aspartate transaminase,30.8%of patients showed alanine transaminase,and 30%of patients showed lactate dehydrogenase.These results provide basic information for clinical identification and prevention of bjnv infection.In addition,the expression vector of BJNV-N protein was successfully constructed,and protein size was 62 KD.The positive rate of IgM was 62.7%(42/67),which indicated that the 42 patients were just infected with BJNV,and the positive rate of IgG was 29.9%(20/67),which indicated that the 20 positive patients had been infected with BJNV before or were in the stage of persistent infection.5.We investigated the prevalence of BJNV in different areas and animals.The results showed that the virus could be detected in ticks,cattle and sheep,including six kinds of ticks(the total positive rate was 1.5%):Dermacentor silvarum(2.1%),Ixodes crenulatus(2.1%),Ixodes persulcatus(2.0%),Haemaphysalis longicornis(1.1%),Haemaphysalis conicinna(1.1%)and Dermacentor nuttalli(0.4%),indicating multifarious ticks are susceptible hosts of BJNV.There was no significant difference in the total infection rate of BJNV among different animals(P=0.56).The total positive rate of BJNV in ticks from different provinces was significantly different(P=0.003).Inner Mongolia was the highest(4.3%),followed by Jilin Province(3.3%)and Heilongjiang Province(1.0%),indicating that there was no geographical limit for the BJNV transmission.There was significant difference in the positive rate of BJNV in Jilin Province(P<0.001).I.crenulatus was the main vector-tick species of BJNV in Jilin Province,and there was significant difference in the positive rate of bjnv virus among 3 species of ticks in Inner Mongolia(P<0.001).I.persulcatus was the main tick species BJNV in Alongshan area of Inner Mongolia,which indicated that the vector-tick species of BJNV in different provinces are different.A total of 441 cattles and sheep serum samples were collected in Inner Mongolia,including 213 cattles and228 sheep samples for molecular and serological screening.The samples of cattle and sheep were deetecte with SYBR fluorescence-quantitative q PCR method.The results showed that the total positive rate of sheep was 55.3%(118/213,95%CI:47.7–61.4),and the total positive rate of cattle was 37.7%(86/228,95%CI:34.4–44.4).There was no significant difference between the two methods(P>0.05).By quantifying the copy number of BJNV,it was found that there was a significant difference in the copy number of BJNV between cattle and sheep(P<0.01).The positive rate of ELISA was28.2%(60/213)in sheep and 19.7%(45/228)in cattle.Molecular and serological methods showed that cattle and sheep as mammals were susceptible to BJNV.The amino acid phylogenetic tree of L and S segments of BJNV from different hosts showed that 18 strains of BJNV from different hosts were all classified into Nairovirus-like family of Bunyaviridae,which was in the same branch as BJNV HLJ2015 and GTV.The amino acid homology of L and S segments from 18 strains of tick from different hosts,tick-bitten patients,cattle and sheep in tick natural foci was more than 98%,which indicated that BJNV existed in tick,livestock and human,and the disease caused by BJNV was confirmed to be a novel zoonosis.This study revealed a novel virus in Nairovirus-like genus in Bunyaviridae infecting human.It proves that Nairovirus-like genus can infect human and widely spread in tick-biten patiens at first in the world,which has high public health significance.At the same time,BJNV is susceptible to a variety of tick species,and the transmission of BJNV is not limited by geographical scope.The main tick species have different transmission vectors among different provinces.And mammals,such as cattle and sheep are host.The disedase infected by BJNV was a novel zoonosis. |
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