| Objective: As the change of global climate,ecological environment and increase of human activities,pathogens circulating in nature spill over to human living environment more frequently,which leads to natural focus disease.Natural focus disease is the main source of emerging infectious disease,which is huge threat to human lives and development.To provide knowledge for more effective methods of prevention and treatment,this research investigated epidemiological,clinical and genetic evolutionary characteristics of severe fever with thrombocytopenia syndrome(SFTS)and coronavirus disease 2019(COVID-19),which were important in China.To maintain health of citizens on the islands in southeast coast areas,pathogens related to natural focus diseases need to be detected,through methods like high-throughput sequencing technology.Methods:Part 1 Generalized additive model(GAM)was used to evaluate the influence of meteorological factors and SFTS.Spatial information technology was used to discover the relationship between geographical environment and SFTS.Samples of wild animals in epidemic areas were collected to find potential hosts.Clinical data were collected in Zhejiang and Henan Provinces.Independent t-test,Cox regression model,and generalized estimating equation(GEE)were applied to identify clinical characteristics of SFTS.Pathogen diagnosis and viral loads detection were completed through quantitative real-time PCR.Specific antibody of Ig M were detected through ELISA methods.Viruses from SFTS patients were isolated from serum after cell infection,virus culture and identification of virus.Isolated viruses were then sequenced with designed primers.Phylogenetic analysis was used to characterize evolutionary history of SFTS virus.Part 2 We collected information on demographic and temporal characteristics,exposure history,and laboratory examinations of all laboratory-confirmed COVID-19 patients that had been admitted to the 5th Medical Center of Chinese PLA General Hospital.Cox regression analysis was applied to identify factors that predicted disease progression.With the use of available public data,we conducted recombination analysis to detect potential recombination events.Haplotype network analysis was performed to depict the evolutionary characteristics of SARS-Co V-2.Affinity changes were predicted to assess the effect of viral mutations occurring in receptor-bind region of the spike protein.Part 3 Blood,tissues,throat swabs and anal swabs of rodents,bats and birds were collected around living and working areas on southeast coastal islands.Nucleic acid of lung tissues was extracted,controlled in quality,sequenced,assembled and identified with bioinformatics tools and reference virus sequence in public database.Results:Part 1.A total of 136 SFTS patients in Zhoushan and Ningbo and 1574 SFTS patients were included.In general,dead patients were elder than survival patients(P<0.001).No significant difference in gender and days from onset to admission was observed between two groups.In Zhejiang Province,prevalent onset time was summer(June to September).On Daishan islands,most viruses derived from patients were in genotype E(P =0.002).Local rainfall,sunshine duration and atmospheric temperature were significantly related to incidence rate(P<0.05).Higher frequency of myalgia and fatigue appeared in survival patients in Zhejiang(P < 0.05),while higher frequency of respiratory symptoms,neurological symptoms and hemorrhage symptoms were observed in dead patients in Henan(P<0.05).Multivariate Cox regression analysis indicated that high blood urea nitrogen(BUN)was an independent risk factor for progression of SFTS in Zhejiang and fatigue symptom was protective factor,with hazard ratio(95% confidence interval)of 1.073(1.021-1.128)and 0.190(0.053-0.675).For all patients at admission,laboratory examinations of lactate dehydrogenase(LDH),glutamic oxalacetic transaminase(AST),BUN and creatinine(CREA)had higher values in dead patients(P≤0.001).Dynamic trends of laboratory examinations and viral loads were different between dead and survival patients(PGEE<0.05),except items of white blood cells(WBC)in Zhejiang and WBC and creatine kinase(CK)in Henan.Dynamic trend of alanine aminotransferase(ALT)was different between recovered patients of Zhejiang and Henan,while trend of CREA was different between dead patients of two provinces(PGEE<0.001).A total of 90 rodents were collected in Zhoushan,with negative result of SFTSV detection.Viruses isolated from 174 patients were sequenced.All strains(117/117)in Henan were in genotype A,while most strains in Zhoushan(52/57)and Ningbo(14/25)were in genotype E.Segmental recombination was observed(9 strains in Zhoushan and 1 strain in Ningbo).Evolutionary analysis indicated that SFTSV originated from Henan,transmitting into Zhejiang Province and South Korea in 1990-2005 from Jiangsu Province.In 2005-2012,viruses transmitted into Japan and back to Zhejiang Province form Korea.Part 2A total of 55 consecutive COVID-19 patients were characterized in this study.The mean incubation period of COVID-19 was 8.42 days.Asymptomatic carriers might transmit SARS-Co V-2.The course of COVID-19 is approximately 2 weeks.Compared with patients without pneumonia,those with pneumonia were 15 years older(P<0.001),had a higher prevalence of hypertension(32.4% vs.0%,P=0.004),higher frequencies of having fever(94.1% vs.71.4%,P=0.043)and cough(61.8% vs.33.3%,P=0.040),higher levels of interleukin-6(14.61 vs.8.06pg/m L,P=0.040),B lymphocyte proportion(13.0% vs.10.0%,P=0.024),erythrocyte sedimentation rate(17.00 vs.6.00m/60 min,P=0.006),low account(<190/μL)of CD8+ T cells(33.3% vs.0,P=0.019).The daily values of T lymphocyte counts and CD8+ T lymphocyte counts were higher in patients without pneumonia than in patients with pneumonia within 20 days after illness onset and the gap became larger gradually,which indicated that CD8+ T cell-mediated immunity needs a long time to recover.Sequence similarity between coronaviruses(Co Vs)from pangolin and SARS-Co V-2 was lower than that between SARS-Co V-2 and Bat-Ra TG13(Co V from bats).Our haplotype network analysis indicated that SARS-Co V-2 strains identified worldwide were grouped into 5 clusters(A to E).The SARS-Co V-2 strains isolated in Wuhan,China,were linked to cluster C,whereas those isolated in USA covered all the 5 clusters.Mutations V367 F and N354 D at the receptor binding domain of the spike protein increased the receptor-binding affinity of SARS-Co V-2.Part 3A total of 44 animal samples were collected Daishan Island of Zhoushan,which located near the East China Sea.Viruses of Siphoviridae,Polyomaviridae and Retroviridae were identified after high throughput sequencing.On an island of the South China Sea,a total of 16 rodent samples and 31 bird samples were collected.The average rat density among all investigation sites was 3.7%,with highest density of 10%.Various kinds of birds were observed on the island.Conclusion:Spatiotemporal distribution of SFTS was related to meteorological and geographical factors.Clinical characteristics of SFTS in Zhejiang was similar to that of Henan,but differences were observed.Appropriate measures of prevention and treatment should be taken according to these results.Phylogenetic analysis provided evidence that SFTS pathogens transmitted from Zhejiang to South Korea and Japan.It was helpful for discovering natural hosts in the future.Various kinds of viruses were identified from animal samples,which were collected from Zhoushan.Those viruses were not reported in Zhoushan before.Higher species richness of animals on island of South China Sea was observed.According to the results,attention should be paid on prevention of natural-focus diseases.COVID-19 distributed outside Hubei is mostly related to exposure to Wuhan,but hospital-mediated transmission and asymptomatic carrier-mediated transmission might occur in elders with underlying diseases and sensitive recipients.SARS-Co V-2-caused lymphocytoponia facilitates the development and progression of pneumonia.The outcomes of this study should be referenced for the prevention and control of COVID-19 outside the outbreak zone.Wuhan is not the initiating place of the Co Vs spillover from natural reservoirs to humans.V367 F and N354 D might facilitate the transmission of SARS-Co V-2. |
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