Hepatitis C virus genotype diversity in Ningxia Hui autonomous region, Northwestern China

Abstract Hepatitis C virus (HCV) genotypes vary greatly in different regions. The aim of this study is to investigate the distribution of HCV genotypes in HCV infected patients, in Ningxia Hui Autonomous Region. Nucleic acid extraction and amplification were performed with test kits on 153 HCV infected patients serum samples. The HCV viral load was measured using reverse transcriptase PCR (RT-PCR) and HCV genotypes were determined. Among the 153 HCV-infected patients, 56 had genotype (GT)1b (36.60%), 45 had GT2a (29.40%), 23 had GT3a (15.00%), 14 had GT3b (9.20%),13 had GT6a (8.50%), 1 had GT1g (0.70%), 1 had GT6xa (0.70%). In GT1b, 21.40% were female and 78.60% were male; in GT2a, 42.20% were female and 57.80% were male;Males were most prevalent in genotypes 1b(39.30%), while female were most prevalent in genotype 2a(46.30%). Rare GT1g and GT6xa were also detected in males. The 41-50 year age group had the highest HCV prevalence of 32.00%. HCV GT1b is the predominant HCV genotype in Ningxia Hui Autonomous Region.


Introduction
The World Health Organization (WHO)'s global hepatitis strategy aims to reduce new hepatitis infections by 90% and deaths by 65% between 2016 and 2030.Hepatitis C virus (HCV) belong to the Hepacivirus genus, with six major genotypes, which can cause liver disease (Lindenbach and Rice, 2005;Bilal, et al., 2022).Determination of HCV genotypes and subtypes are important to predict response to antiviral therapy for the efficacy and barrier to resistance of non-pangenotypic direct-acting antiviral agents (DAAs) depend on the HCV genotypes and subtypes (Hayes, et al., 2022).In developing countries like China, identification of HCV genotypes and subtypes is still lacking.The overall HCV treatment can be improved by genotyping and quantification of HCV virus, which could also be used as a good indicator for decision making of an optimal treatment regimen.
HCV genotype distribution varies in different regions (Ullah et al., 2021;Petruzziello et al., 2016;Ju et al., 2015;Sallam et al., 2019).So far, there is little literature on the distribution of HCV genotypes in Northwestern China, especially the epidemiology of different age and gender group.Herein, we invesitigated the genotype distribution of HCV-infected patients from Ningxia Hui Autonomous Region, Northwestern China, which can provide a basis found rare subtypes 1g ( 0.65% ) and 6xa ( 0.65% ) by PCR and sequencing (Figure 1).
HCV 1b was the most prevelant genotype in Ningxia Hui Autonomous Region and all of the other areas in China (Table 1).
The highest number of HCV infection was between the age of 41-50 years ( 32.00%).HCV genotype 1b, 3a, 3b and 6a was most common in 41-50 age group ( 40.60%); for further study of HCV prevalence in the region and also guidance for individualized treatment.

Materials and Methods
A total of 153 HCV RNA positive serum samples from 2019 to 2021 enrolled in the further HCV genotyping which were obtained from individuals who were diagnosed as chronic hepatitis C and conducted relevant inspections at the Department of Laboratory Medicine, People's Hospital of Ningxia Hui Autonomous Region, China.The study was approved by the Ethics Committee of the People 's Hospital of Ningxia Hui Autonomous Region.Written consent were all acquired from the patients.The baseline data including age, gender, and prior surgical history were collected from Laboratory Information System (LIS) and the Hospital Management Information System (HIS).
For high precision HCV-RNA quantitative detection (PCR-Fluorescence Probing) 153 enrolled serum samples were processed using Rotor-Gene Q/Thermo-optical Analyzer and supporting diagnostic kit.Data were automatically analyzed by Rotor-Gene Q software.All the above procedures were strictly conducted in accordance with the reagent and instrument instructions.
For HCV genotype and subtype analysis, the HCV RNA was extracted by relevant HCV genotyping kit (PCRfluorescence probe method), Da AN Gene Biotechnology Co, Ltd, Shenzhen, China.The HCV Genotyping Kit, with sensitivity of 5.0×10 3 IU/mL, a region within the HCV-RNA 5′UTR was amplified and identified the five most common HCV genotypes (1b, 2a, 3a, 3b, 6a).The extracted target genes were amplified by T100 Thermal Cycler (Bio-Rad, Hercules, California, USA).Amplification was performed according to the following program conditions: 50°C for 25 minutes, 95°C for 15 minutes, 94°C for 30 seconds, 55°C for 40 seconds, 72°C for 45 seconds, 45 cycles.After the reverse transcription-PCR completed,HCV genotypes and subtype were determined in accordance with the relevant instructions.In addition, the unascertained HCV genotypes and subtypes were sequenced using the Diagnostic Kit For HCV Genotyping (PCR-sequencing Analysis) (Life-DaAn Diagnostic Products Technology Co., Ltd.) and Applied Biosystems 3730 gene sequencer.The sequencing results were analyzed using Chromas software 2.6.6 (Technolysium Ltd.).
The distribution of the HCV genotype and the average viral load were calculated using Microsoft Excel.No additional statistical tests were performed, due to the small sample size.
We also found that the constituent ratio of HCV genotypes ( 1b, 2a, 3a, 3b, 6a, 6xa, 1g ) in Han patients with HCV infection was higher than that in Hui patients(Figure 3).

Discussion
HCV infection causes a major threat to the global public health (Mohd et al., 2013).It is estimated that there were 10 million HCV-infected people in China in 2019.The distribution of HCV genotype and subtype have obvious geographical and population differences around the world.Different HCV genotypes and subtypes respond differently to specific treatment regimens.It is important to clarify the prevalenct HCV genotype in Ningxia Hui Autonomous Region, Northwestern China since few data are currently available concerning the distribution of HCV in Northwestern China.
The changes of HCV genotype/subtype and viral load have important guidance value for optimal clinical treatment.Experts recommend that HCV infected persons should be genotyped before starting antiviral treatment, mainly considering that its affordability in China is better than pan-genotyping scheme.Previously, In China, HCV 1b  (56.80%) was the predominant HCV genotypes, followed by HCV genotype 2, 3, 6.Additionally, HCV genotype 4 and 5 were rare among the whole of China.While in southern and western China, genotype 1b was the main type, and genotype 2, 3 and 6 also accounted for a large proportion.
In genotype 3, the prevalence of genotype 3b was higher than that of 3a.Genotype 3 and 6 HCV infection exists nationwide and no longer restricted to southern and western China (Ju et al., 2015).It is worth noting that the epidemic pattern of HCV in China is evolving due to the change of population mobility.
Our study found that in northwestern China the most common subtype was 1b ( 36.60% ), followed by 2a ( 29.41% ), 3a ( 15.03% ), 3b ( 9.15% ), and 6a ( 8.51% ).That is slightly different from other areas in China.The proportion of HCV genotype 1b (36.60%) in Ningxia Hui Autonomous Region was lower than that in Sichuan Area(76.10%) and Liaoning area(45.10%)(Li et al., 2021;Hu et al., 2021), but the proportion of 3a (15.03%) was higher than these areas.In addition, we found HCV genotype 1g ( 0.65% ) and 6xa ( 0.65% ) by PCR and sequencing, which were rare in Northwestern China.Ningxia Hui Autonomous Region is an underdeveloped inland region in northwestern China.Diversity of HCV gene subtypes in the region brings new challenges for the prevention and control of HCV and other various infectious diseases.In addition, there were few studies on HCV genotype distribution in northwestern China.Therefore, our study will play a key role in the diagnosis, treatment and prognosis of HCV in Ningxia Hui Autonomous Region.
In every group of our study, the incidence in males was higher than in females, and 95.40% of all positive cases were aged over 30 years, mostly between 41-50 years of age (32.00%).The economic development in Ningxia Hui Autonomous Region was unbalanced, people knew less about hepatitis C. The onset of hepatitis C is insidious and chronic, and about 20.00% of patients with hepatitis C can selfheal, mainly because these patients have strong autoimmune function and the amount of infected virus is relatively small.
The viral load of HCV is related to its genotype, and the viral load are of great value for guiding clinical treatment.The mean viral load among patients with HCV genotype 1 infected was reported to be higher than that of infected with genotype 4 (Mishra et al., 2020).However, our research shows that the HCV RNA level of HCV-3a patients is higher than that of other genotypes (Table 2).Previously, Brady and Stoykova (2019) reported that the mean viral load level with HCV genotype 3 is higher than that of others (Brady and Stoykova, 2019).We think that these different research results may be caused by the heterogeneity of the populations studied.
Our research shows that in northwestern China, Ningxia Hui Autonomous Region, the constituent ratio of all the detcted HCV genotypes ( 1b, 2a, 3a, 3b, 6a, 6xa, 1g ) in Han chronic hepatitis C patients were higher than that in Hui patients.We thought that of the Chinese population, 64.05% are of Han nationality and 35.04% are of Hui nationality in Ningxia Hui Autonomous Region, which accounts for the results as mentioned above.Additionally, we found that HCV genotypes in the Hui chronic hepatitis C patients were diverse.In contrary, Li et al.' s reported that the genotype of hepatitis C in Liaoning Province, the Hui nationality,only 3 genotypes were prevalent for Hui patients: GT1b, GT2, and GT3a (Li et al., 2021).Our results were slightly inconsistent with the study of Li et al for there were few amounts Hui chronic hepatitis C patients in Liaoning province.In addition, it is also possible that the results were slightly different due to geographical differences.Liaoning is located in the northeast of China, while Ningxia is located in the northwest of China.

Conclusion
This research reveals the current HCV genotype distribution in Ningxia Hui Autonomous Region, Northwestern China.The results provide a basis for further studying the epidemic situation of hepatitis C virus in Ningxia Hui Autonomous Region and developing targeted prevention and control strategies.It is necessary to conduct extensive research on the distribution of HCV genotypes in Ningxia Hui Autonomous Region for against HCV infection in Ningxia Hui Autonomous Region.

Figure 1 .
Figure 1.Distribution of HCV genotypes in Northwestern China.

Figure 2 .
Figure 2. Proportion of HCV genotypes by gender in Northwestern China.

Figure 3 .
Figure 3. Distribution of HCV genotypes in Han and Hui ethnic groups patients with hepatitis C in Northwestern China.

Table 1 .
HCV genotype distribution in different regions of China.

Table 2 .
HCV genotypes and viral load in different age groups.