| Hepatitis C is a disease mainly transmitted by blood caused by hepatitis C virus(HCV),HCV infection is a major cause of chronic hepatitis,liver cirrhosis and even liver cancer,representing a major public health problem.According to statistics,about170～200 million people worldwide are infected with HCV,and HCV-infected people in China account for about 14% of the total global HCV-infected population,and in2016,the World Health Organization(WHO)proposed a new global goal of eliminating viral hepatitis by 2030.In recent years,the globalization of the economy and the crossborder movement of people have increased the risk and opportunity of virus transmission and the exchange of different genetic subtypes of virus,promoting the prevalence and spread of the virus.Yunnan Province is located in the southwestern border of China and borders several countries in Southeast Asia.Due to the particular geographical location,the diversity of ethnic groups,and the use of injecting drugs,Yunnan has become one of the major areas of HCV epidemics and cross-border importation of HCV into Yunnan occurs frequently.However,what are the current epidemiological characteristics of HCV imported from outside Yunnan? What are the differences between the prevalence characteristics of HCV imported from outside Yunnan and local HCV in Yunnan? There are few reports.Therefore,in this paper,we investigated the populations with HCV infection imported from outside Yunnan and local Yunnan,elucidated the epidemiological variation characteristics of genotypic subtypes imported from outside Yunnan and local Yunnan,compared the epidemiological variation characteristics of genotypic subtypes imported from outside Yunnan and local Yunnan,and analyzed the selection pressure,molecular transmission network and drug resistance-associated mutations,which are important for the surveillance and prevention of HCV in Yunnan Province,and are contributing to China to achieve the strategic goal of eliminating hepatitis C by 2030 as proposed by WHO.First,we collected 520 positive HCV antibody samples imported from abroad to Yunnan from 2017-2019,of which 52.1% were from Southeast Asia and a small number were from Europe,America,northern Asia,South Asia and Africa.We amplified and sequenced 381 and 455 cases for the E1 region and NS5 B region,respectively,constructed a phylogenetic evolutionary tree,and genotyped 511 samples successfully based on the results of the tree construction.The results showed that subtype 3b accounted for 25.24%(129/511),6n and 1b accounted for 15.66%(80/511)and 11.55%(59/511),respectively,6xa accounted for 8.61%(44/511),3a accounted for 8.22%(42/511),1a accounted for 7.44%(38/511),and the unclassified 6 subtypes 5.38%(28/511)and mixed infections 4.31%(22/511).During the three years 2017-2019,GT3 was in a state of relatively stable growth,with relatively small percentages of subtypes2 a and 2b,subtype 2a increasing and then decreasing and fluctuating between 2.19%and 6.75%,subtype 1a showing a decreasing trend,subtype 1b showing some upward trend,and an overall increase in type 6 and mixed infections.Near-full-length genomic amplification of unclassified subtype 6 samples and full-length phylogenetic evolutionary tree analysis showed that one case was separately clustered with the submitted sequences KC844039 and LC368421,and the other three cases were separately clustered;these sequences did not recombine with other classified subtype 6,and there was no epidemiological linkage between the sequences,and the calculated genetic distance also showed compliance with the new subtype nomenclature Resistance analysis of the NS5 B region in 455 typed samples showed that 9.8%(50/455)of patients with type 1b had mutations in C316N/H and 0.4%(2/455)of patients with type 1a had mutations in C316Y/W,all of which were resistant to dasabuvir;one case of type 1b and four cases of type 3b had mutations in C316Y/W.mutation in S282 T in type 1b and four in type 3b patients,a mutation in S282 R in one 6xa patient,and a combined mutation in S282 T and V321 G in one type 3a patient,all of which were strongly resistant to sofosbuvir.Second,based on the number of HCV samples imported from abroad,we randomly collected a total of 500 HCV antibody-positive samples in Yunnan from2017-2019,amplified and sequenced 374 and 371 cases of E1 region and NS5 B region,respectively,constructed a phylogenetic evolutionary tree,and genotyped 445 samples successfully based on the tree construction results.The results showed that subtype 3b accounted for the largest proportion of 39.8%(177/445),followed by 2a with 17.5%(78/445),1b with 12.6%(56/445),3a with 12.6%(56/445),6n with 9.7%(43/445)and6a with 4.3%(19/445).For the three years from 2017-2019,the GT3 accounted for the first place all the time,GT1 had a stable decreasing trend,GT2 increased and then decreased showing fluctuating changes,and GT6 type and mixed infections increased overall.In the resistance analysis of the NS5 B region of the typed 371 samples,13.2%(49/371)of patients with type 1b had a mutation in C316 N that produced resistant resistance to dasabuvir,and mutations in S282 T with strong resistance to sofosbuvir were found in five patients with type 3b.In addition,1.1%(4/371)were detected in subtypes 2a and 3b,respectively mutations in S289 L and 0.3%(1/371)in V321 A with resistant resistance to sofosbuvir.Finally,we compared the prevalence change characteristics of local and overseas imported HCV genotypes/subtypes in Yunnan from 2017-2019,and the results showed significant differences in the prevalence of multiple genetic subtypes during 2017-2019,finding highly significant differences in the prevalence of subtypes 1a,2a,6xa,and unclassified 6(p<0.001),subtypes 3a,6n,6v,6xj and mixed infections had significant differences in prevalence(p<0.05);the proportion of local Yunnan subtypes 2a,3a,and3 b were consistently higher than those imported from abroad in these three years,in contrast,subtypes 1a and 6 were more prevalent in the imported from abroad,and subtype 2b was only found in the imported from abroad population in 2017.A comparative analysis of the selection pressure on the major genotypes of HCV in Yunnan and those imported from abroad showed that,except for subtype 6a,the positive selection sites of the E1 region of HCV in Yunnan differed from those imported from abroad,with more positive selection sites for subtypes 3a and 6n in Yunnan compared to those imported from abroad,and the opposite for subtype 3b.In addition,we performed molecular transmission network analysis of local and imported HCV from Yunnan,and the results showed that there were clusters formed by imported sequences from abroad and local sequences from Yunnan,and there might be cross-border transmission relationship.The results showed that a high proportion of C316 N mutations in subtype 1b were found to be resistant to dasabuvir,and a small proportion of patients with subtype 3b were found to have strong resistance to sofosbuvir with mutations in S282 T,and Only a few genetic subtypes with different site of drug resistance mutations occurred.In summary,this paper elucidates the molecular epidemiological characteristics of HCV imported from outside Yunnan,identifies and names two new GT6 subtypes 6xk and 6xl for the first time,characterizes the molecular epidemiological characteristics of local HCV in Yunnan,and compares the molecular epidemiological characteristics of local and imported HCV in Yunnan,and compares the positive selection sites,molecular transmission networks and drug resistance-associated mutations in the two populations.The analysis provided insights into the development of treatment and prevention strategies for HCV in Yunnan and the spread of HCV to surrounding areas. |
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