Infection, Genetics and Evolution 73 (2019) 139–145 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Research paper Complicated HCV subtype expansion among drug users in Guangdong province, China T ⁎ Jin Yana, , Xiao-Bing Fua, Ping-Ping Zhoub, Xiang Heb, Jun Liua, Xu-He Huangb, Guo-Long Yua, Xin-Ge Yana, Jian-Rong Lia, Yan Lia, Peng Lina a Guangdong Provincial Center for Disease Control and Prevention, 160 Qunxian Road, Panyu District, Guangzhou 511430, Guangdong, China b Guangdong Provincial Institute of Public Health, Guangdong Provincial Center for Disease Control and Prevention, 160 Qunxian Road, Panyu District, Guangzhou 511430, Guangdong, China ARTICLE INFO ABSTRACT Keywords: Guangdong Province is one of the most developed and populous provinces in southern China. The subtype HCV situation of hepatitis C virus (HCV) in Guangdong remains unknown. The aim of this study was to investigate Subtype and estimate the HCV subtypes in drug users (DU) using a city-based sampling strategy to better understand the Drug users characteristics of HCV transmission in Guangdong. Archived plasma samples (n = 1074) from DU who were Molecular epidemiology anti-HCV positive in 2014 were selected randomly from 20 cities in Guangdong Province. Subtypes were de- Guangdong termined based on core and/or E1 sequences using phylogenetic analysis. The distributions of HCV subtypes in DU and different regions were analyzed. A total of 8 genotypes were identified. The three main HCV subtypes in DU in Guangdong were 6a (63.0%), 3a (15.2%), and 3b (11.8%). Significant differences were discovered among different registered residency and regions but not among genders, marital status, education level, or drug use patterns. HCV subtype 3b was significantly higher in Guangdong residents than in non-Guangdong residents. In contrast, HCV subtype 6a was significantly lower in Guangdong residents than in non-Guangdong residents. Subtype 1b in eastern Guangdong (eastern) was significantly lower, while 6a was significantly higher when compared with other regions. Subtype 3a in the Pearl River Delta (PRD) region was significantly higher, while 3b was significantly lower when compared with other regions. In western Guangdong, HCV subtype 3a was sig- nificantly lower when compared with other regions. Additionally, in northern Guangdong subtypes 1b and 3b were significantly higher, while 6a was significantly lower when compared with other regions. Our study re- vealed the diversity and distribution of HCV subtypes in DU in nearly all the cities in Guangdong. The results provide essential information that will allow the establishment of specific intervention strategies that may help prevent HCV transmission. 1. Introduction virus (HCV) incidence in different groups in Guangdong. However, information regarding drug users (DU) from DU sentinel surveillance Guangdong Province, located on the southern coast, is one of the sites was available. According to a report in 2016, the average Anti- most developed provinces in China and has the largest population and HCV positive rate in DU in Guangdong Province was 46.51% (Huang the highest population density. Moreover, the transient population ac- et al., 2016), which was much higher than the HCV infection rate in counts for 17% of the total population. The number of annually re- Chinese blood donors (0.34%) (Fu et al., 2010) and in the general po- ported cases of hepatitis C in Guangdong Province has been increasing pulation of Guangdong Province (2.25%) (Luo et al., 2005). Due to the for several years since 2005. For this reason Guangdong Province was lack of large-scale HCV molecular epidemiological studies, the dis- ranked top for the most number of hepatitis C cases in China in 2017 tribution of HCV subtypes is not yet clear. Studies found that the main (Fu et al., 2015) Owing to the lack of epidemiological information for subtypes of clinical patients in Guangdong Province were 1b, 6a, and 2a reported hepatitis C cases, it is impossible to understand the hepatitis C (Huang et al., 2018; Yuan et al., 2017) while 1b, 6a, 3b were common Abbreviations: HCV, Hepatitis C virus; DU, Drug users; IDU, Injecting drug user; NIDU, Non- Injecting drug user; NA, Not available; χ2, Chi squared test; PRD region, Pearl River Delta region ⁎ Corresponding author at: Institute of HIV/AIDS Control and Prevention, Guangdong Provincial Center for Diseases Control and Prevention, RM. 2530, 160 Qunxian Road, Panyu District, Guangzhou 511430, Guangdong, China. E-mail addresses: [email protected], [email protected] (J. Yan). https://doi.org/10.1016/j.meegid.2019.04.031 Received 12 March 2019; Received in revised form 24 April 2019; Accepted 27 April 2019 Available online 30 April 2019 1567-1348/ © 2019 Published by Elsevier B.V. J. Yan, et al. Infection, Genetics and Evolution 73 (2019) 139–145 in blood donors (Rong et al., 2014). The most common subtypes in DU Neighbor-joining phylogenetic trees were reconstructed to determine were found to be 6a, 3a, 3b or 6a, 3a, and 1b (Chen et al., 2011; Kuang the subtypes of core and E1 with the Kimura 2-parameter substitution et al., 2015). Interestingly, 1b is the main subtype in the general po- model and evaluated by the bootstrap method with 1000 replicates pulation of Guangdong Province while 6a is the main subtype among using MEGA 6. DU. However, previous studies had their limitations and they do not reveal any insight into the subtype distribution of non-injecting drug 2.4. Estimation of HCV subtypes in cities and regions users (NIDU). In this study, we systematically sampled the DU in the DU sentinel surveillance sites across the province. We estimated the dis- The basic analysis unit in our study was a specific subtype in a tribution of HCV subtypes in the DU in Guangdong using the subtypes prefecture city. The number of cases for each subtype in a city was of the 1074 archived samples combined with their epidemiological estimated by multiplying the proportion of that genotype in that city to information in the prefecture-level cities in Guangdong. The subtypes the total number of anti-HCV positive cases in that same city. The and the epidemiology of injecting drug users (IDU) and NIDU were also corrected total numbers and corresponding sampling ratios for each city fl compared. Our results provide a comprehensive dataset re ecting the are listed in Table 2. The estimated number of each subtype in each characteristics and diversity of HCV in DU in Guangdong. region across the province was obtained by summing the units across all cities. Since no samples were acquired from the city of Heyuan, only 20 2. Material and methods cities were included in calculation. 2.1. Study participants and sampling strategy 2.5. Statistical analysis All the specimens were derived from participants in 38 DU sentinel Statistical analysis was performed using SPSS (Ver. 23.0) and a P surveillance sites, including 17 in the PRD, 7 in the eastern, 8 in the value of < 0.05 was considered to be statistically significant. western, and 6 in northern Guangdong Province. In 2014, a total of Differences between regions were studied using the corrected number 16,133 DU were monitored in Guangdong Province, of which 7703 of anti-HCV positive cases and subtypes. cases were anti-HCV positive. A minimum sample size was calculated according to the anti-HCV positive number of each city in 2014 3. Results (Table 2). A total of 1074 plasma specimens were randomly selected from the 7703 anti-HCV positive cases from 20 prefecture cities except 3.1. Distribution of HCV subtypes among DU in Guangdong Heyuan. A questionnaire survey was conducted by all participants and their general demographics, sexual behavior and other relevant in- A total of 1074 samples were selected from the anti-HCV positive formation were collected. cases in this study, accounting for 13.8% of anti-HCV positive cases in all DU sentinel surveillance sites (except Heyuan). A total of 976 cases 2.2. RNA extraction, amplification, and sequencing were determined for the HCV subtypes, of which 939 Core (87.4%) subtypes and 837 (77.9%) E1 subtypes were acquired, and 800 samples Viral RNA was extracted from 200 μl of plasma using the MagPure (74.5%) were acquired for both subtypes (Table 2). Viral Nucleic Acid KF Kit (Magen, Guangzhou, China) with a KingFisher The subtype distributions are show in Fig. 1 and Table 3. Phyloge- Flex system (Thermo Fisher Scientific, Waltham, MA, USA) following netic trees of the core (Fig. 2A) and E1 (Fig. 2B) sequences show that the manufacturer's instructions. Both core (H77: 337–697 nt) and E1 eight HCV subtypes were identified in this study, namely 1a, 1b, 3a, 3b, (H77: 732–1322 nt) fragments were amplified with PrimeScript One 6a, 6e, 6u, and 6n. The predominant subtypes circulating in Guangdong Step RT-PCR Kit and Premix Taq (Takara Bio, Dalian, China). The core are 6a (63.0%), 3a (15.2%), 3b (11.8%) and 1b (6.7%). Minor HCV fragment was amplified using the primers and amplification conditions subtypes included 1a, 6e, 6n, and 6u. These were classified as “other” as previously described (Lu et al., 2005). The E1 fragment was ampli- subtypes. fied using the primers as described above first，then the samples that fi had been failed were ampli ed with in-house degenerate primers 3.2. Distribution of HCV subtype by geographic region (Table 1) under the following conditions: annealing at 50 °C for 5 cycles fi then at 54 °C for 25 cycles for the rst round and 58 °C for 5 cycles, then Guangdong Province can be classified into four regions: the PRD, 62 °C for 25 cycles for the second round. eastern, western, and northern regions.