HCV subtype characterization among injection drug users: implication for a crucial role of Zhenjiang in HCV transmission in China

Background

HCV transmission is closely associated with drug-trafficking routes in China. However, the transmission route of HCV in Eastern China remains unclear. Here, we investigate the role of Zhenjiang city of Jiangsu province, an important transportation hub linking Shanghai with other regions of China, in HCV transmission.

Methodology/Principal Findings

A total of 141 whole blood samples were collected from injection drug users (IDUs) in Zhenjiang and then tested for HCV infection. Of them, 115 HCV positive plasmas were subjected to RNA extraction, RT-PCR amplification, and sequencing. The subtype characterization and the evolutionary origin of HCV strains circulating in Zhenjiang were determined using polygenetic or phylogeographic analyses. Seven HCV subtypes 1b, 2a, 3a, 3b, 6a, 6e and 6n were detected among Zhenjiang IDUs, showing a complex HCV epidemic. The most predominant subtypes were 3a (38%) and 1b (26.8%). Among these subtypes, subtypes 3b, 6n and 6e originated from Southwestern China (i.e., Yunnan and/or Guangxi), subtypes 2a and 6a from Southern China (i.e., Guangdong), subtype 1b from Central (i.e., Henan) and Northwestern (i.e., Xinjiang) China, and subtype 3a from Southwestern (i.e., Yunnan) and Northwestern (i.e., Xinjiang) China. From Zhenjiang, subtypes 1b and 2a were further spread to Eastern (i.e., Shanghai) and Northern (i.e., Beijing) China, respectively.

Conclusions/Significance

The mixing of seven HCV subtypes in Zhenjiang from all quarters of China indicates that as an important middle station, Zhenjiang plays a crucial role in HCV transmission, just as it is important in population migration between other regions of China and Eastern China.     

HCV Genotypes,Characterization of Mutations Conferring Drug Resistance to Protease Inhibitors,and Risk Factors among Blood Donors in S?o Paulo,Brazil

Background

Hepatitis C virus (HCV) infection is a global health problem estimated to affect almost 200 million people worldwide. The aim of this study is to analyze the subtypes and existence of variants resistant to protease inhibitors and their association with potential HCV risk factors among blood donors in Brazil.

Methods

Repeat anti-HCV reactive blood donors are systematically asked to return for retest, notification, and counseling in which they are interviewed for risk factors for transfusion-transmitted diseases. We analyzed 202 donors who returned for counseling from 2007 to 2010 and presented enzyme immunoassay- and immunoblot-reactive results. The HCV genotypes and resistance mutation analyses were determined by the direct sequencing of the NS5b and NS3 regions, respectively. The HCV viral load was determined using an in-house real-time PCR assay targeting the 5′-NCR.

Results

HCV subtypes 1b, 1a, and 3a were found in 45.5%, 32.0%, and 18.0% of the donors, respectively. The mean viral load of genotype 1 was significantly higher than that of the genotype 3 isolates. Subtype 1a was more frequent among young donors and 3a was more frequent among older donors. Protease inhibitor-resistant variants were detected in 12.8% of the sequenced samples belonging to genotype 1, and a higher frequency was observed among subtype 1a (20%) in comparison to 1b (8%). There was no difference in the prevalence of HCV risk factors among the genotypes or drug-resistant variants.

Conclusions

We found a predominance of subtype 1b, with an increase in the frequency of subtype 1a, in young subjects. Mutations conferring resistance to NS3 inhibitors were frequent in treatment-naïve blood donors, particularly those infected with subtype 1a. These variants were detected in the major viral population of HCV quasispecies, have replicative capacities comparable to nonresistant strains, and could be important for predicting the response to antiviral triple therapy.     

Screening for arboviruses in healthy blood donors: Experience from Karachi,Pakistan

Highlights:
1 Potential transfusion-related transmission of DENV, WNV and JEV was investigated in healthy blood donors from the blood bank of Aga Khan University during July to December 2018.
2 ELISA for DENV, WNV and JEV IgM antibodies and RT-PCR for viral RNA detection were performed.
3 Of the 360 blood donors screened, IgM antibodies for DENV and WNV were positive in 3.9% and JEV in 0.28% respectively while none of the blood donors tested positive for RT-PCR.
4 Majority of the seropositive donors were between 19 to 30 years of age and residents of urban areas, mainly from Karachi City.
5 DENV and WNV seropositivity were significantly associated with residence in Malir District of Karachi.     

Genotype Distribution and Molecular Epidemiology of Hepatitis C Virus in Hubei,Central China

Background

Little is known about the molecular epidemiology of hepatitis C virus (HCV) infection in Central China.

Methodology/Principal Findings

A total of 570 patients from Hubei Province in central China were enrolled. These patients were tested positive for HCV antibody prior to blood transfusion. Among them, 177 were characterized by partial NS5B and/or Core-E1 sequences and classified into five subtypes: 1b, 83.0% (147/177); 2a, 13.0% (23/177); 3b, 2.3% (4/177); 6a, 1.1% (2/177); 3a, 0.6% (1/177). Analysis of genotype-associated risk factors revealed that paid blood donation and transfusion before 1997 were strongly associated with subtypes 1b and 2a, while some subtype 2a cases were also found in individuals with high risk sexual behaviors; subtypes 3b, 6a, and 3a were detected only in intravenous drug users. Phylogeographic analyses based on the coalescent datasets demonstrated that 1b, 2a, 3b, and 6a were locally epidemic in Hubei Province. Among them, subtype 1b Hubei strains may have served as the origins of this subtype in China, and 2a and 3b Hubei strains may have descended from the northwest and southwest of China, respectively, while 6a Hubei strains may have been imported from the central south and southwest.

Conclusion/Significance

The results suggest that the migration patterns of HCV in Hubei are complex and variable among different subtypes. Implementation of mandatory HCV screening before donation has significantly decreased the incidence of transfusion-associated HCV infection since 1997. More attention should be paid to intravenous drug use and unsafe sexual contact, which may have become new risk factors for HCV infection in Hubei Province.     

Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice

Background

With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.

Methodology/Principal Findings

A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.

Conclusions/Significance

In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.     

HCV 6a prevalence in Guangdong province had the origin from Vietnam and recent dissemination to other regions of China: phylogeographic analyses

Background

Recently in China, HCV 6a infection has shown a fast increase among patients and blood donors, possibly due to IDU linked transmission.

Methodology/Findings

We recruited 210 drug users in Shanwei city, Guangdong province. Among them, HCV RNA was detected in 150 (71.4%), both E1 and NS5B genes were sequenced in 136, and 6a genotyped in 70. Of the 6a sequences, most were grouped into three clusters while 23% represent emerging strains. For coalescent analysis, additional 6a sequences were determined among 21 blood donors from Vietnam, 22 donors from 12 provinces of China, and 36 IDUs from Liuzhou City in Guangxi Province. Phylogeographic analyses indicated that Vietnam could be the origin of 6a in China. The Guangxi Province, which borders Vietnam, could be the first region to accept 6a for circulation. Migration from Yunnan, which also borders Vietnam, might be equally important, but it was only detected among IDUs in limited regions. From Guangxi, 6a could have further spread to Guangdong, Yunnan, Hainan, and Hubei provinces. However, evidence showed that only in Guangdong has 6a become a local epidemic, making Guangdong the second source region to disseminate 6a to the other 12 provinces. With a rate of 2.737×10⁻³ (95% CI: 1.792×10⁻³ to 3.745×10⁻³), a Bayesian Skyline Plot was portrayed. It revealed an exponential 6a growth during 1994–1998, while before and after 1994–1998 slow 6a growths were maintained. Concurrently, 1994–1998 corresponded to a period when contaminated blood transfusion was common, which caused many people being infected with HIV and HCV, until the Chinese government outlawed the use of paid blood donations in 1998.

Conclusions/Significance

With an origin from Vietnam, 6a has become a local epidemic in Guangdong Province, where an increasing prevalence has subsequently led to 6a spread to many other regions of China.     

Different HCV Genotype Distributions of HIV-Infected Individuals in Henan and Guangxi,China

Background

Due to shared transmission routes, hepatitis C virus (HCV) infection is highly prevalent among people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is associated with hepatotoxicity, leading to the negative effects on patients with HIV/HCV co-infection. In order to provide valuable information for HCV management in this particular population, we investigated the HCV genotypes in HIV-infected individuals from Henan and Guangxi, the two provinces with the most HIV-infected cases in China.

Methods

Individuals, who acquired HIV infection through various risk routes, were recruited from Henan and Guangxi. Test of antibodies against HCV (anti-HCV) was conducted, and detection of HCV RNA was performed by PCR amplification. HCV subtypes were determined by direct sequencing of amplicons, followed by phylogenetic analysis.

Results

We recruited a total of 1,112 HIV-infected people in this present study. Anti-HCV was detected from 218 (50.1%) patients from Henan and 81 (12.0%) patients from Guangxi, respectively. The highest prevalence of HIV/HCV co-infection was observed from FBDs (former blood donors) (87.2%) in Henan and IDUs (intravenous drug users) (81.8%) in Guangxi, respectively. The seroprevalence rate of HCV among people with sexual contact was significantly higher in Henan than in Guangxi (18.7% vs. 3.5%, P<0.05). The positive rate of HCV RNA in Henan and Guangxi was 30.6% (133/435) and 11.2% (76/677), respectively. Moreover, we found that 20 anti-HCV negative samples were HCV positive by PCR amplification. HCV subtype 1b (52.7%) was predominant in Henan, followed by subtype 2a (41.9%). The most frequently detected subtypes in Guangxi were 6a (35.6%) and 3b (32.9%).

Conclusion

The HCV genotype distributions were different in HIV-infected people from Henan and Guangxi. HIV/HCV co-infection was not only linked to the transmission routes, but also associated with the geographic position.     

Correlation of Viral Loads with HCV Genotypes: Higher Levels of Virus Were Revealed among Blood Donors Infected with 6a Strains

Background

Both HCV genotypes and viral loads are predictors of therapeutic outcomes among patients treated with α-interferon plus ribavirin; however, such correlation has only been studied for genotypes 1, 2, and 3 but not for genotype 6.

Methodology/Findings

299 voluntary blood donors were recruited who were HCV viremic. Their mean age was 31.8; the male/female ratio was 3.82 (225/59). The viral loads of HCV were measured using the COBAS AmpliPrep/COBAS TaqMan test (CAP/CTM) while HCV genotypes were determined by direct sequencing the partial NS5B region. HCV genotypes 1, 2, 3, and 6 were determined in 48.9%, 8.7%, 12.3%, and 30.1% of the donors, respectively, and the levels of mean viral loads in genotype 1 and 6 significantly higher than that of 2 and 3 (P<0.001). As a whole, the viral loads in male donors were higher than in female (P = 0.006). Moreover, the donors'' gender and HCV genotypes are independently correlated with the measured viral loads.

Conclusion

HCV genotype 1 and 6 had significantly higher viral loads than genotype 2 and 3.     

Hepatitis G virus (GBV-C) infection among Brazilian patients with chronic liver disease and blood donors

Background: The recently discovered hepatitis G virus (HGV) belongs, as hepatitis C virus (HCV), to the Flaviviridae family. HGV has been isolated from the serum of patients with non A-E hepatitis. However, the association of HGV with hepatitis is uncertain.Objective: To determine the HGV prevalence in blood donors and in patients with liver disease and to evaluate a possible correlation between HGV infection and liver disease.Study design: Sera from a total of 113 consecutive patients with chronic liver disease were submitted to a series of liver enzymes and function tests and analyzed for the presence of HBsAg, anti-HBs, anti-HBc, anti-HCV, HCV RNA and HGV RNA. Prevalence of HGV RNA was determined in a group of 87 blood donors.Results: Nine (10%) sera from blood donors and 15 (13%) sera from patients with chronic liver disease were HGV RNA positive. Some 28 (25%) patients were HCV RNA positive, with genotypes 1a, 1b and 3 present in 10, 12 and 5 patients, respectively. A total of 20 (18%) patients were HBsAg carriers. Five (4%) patients were double infected (one with HBV+HCV, one with HBV+HGV and three with HCV+HGV).Conclusion: The proportion (10%) of HGV-infected blood donors was very high when compared with other countries. The results did not allow to establish HGV as an etiologic agent for chronic liver disease. The parenteral route was the presumed means of HGV transmission for only one-third of the patients.     

The Evolutionary Rates of HCV Estimated with Subtype 1a and 1b Sequences over the ORF Length and in Different Genomic Regions

Background

Considerable progress has been made in the HCV evolutionary analysis, since the software BEAST was released. However, prior information, especially the prior evolutionary rate, which plays a critical role in BEAST analysis, is always difficult to ascertain due to various uncertainties. Providing a proper prior HCV evolutionary rate is thus of great importance.

Methods/Results

176 full-length sequences of HCV subtype 1a and 144 of 1b were assembled by taking into consideration the balance of the sampling dates and the even dispersion in phylogenetic trees. According to the HCV genomic organization and biological functions, each dataset was partitioned into nine genomic regions and two routinely amplified regions. A uniform prior rate was applied to the BEAST analysis for each region and also the entire ORF. All the obtained posterior rates for 1a are of a magnitude of 10⁻³ substitutions/site/year and in a bell-shaped distribution. Significantly lower rates were estimated for 1b and some of the rate distribution curves resulted in a one-sided truncation, particularly under the exponential model. This indicates that some of the rates for subtype 1b are less accurate, so they were adjusted by including more sequences to improve the temporal structure.

Conclusion

Among the various HCV subtypes and genomic regions, the evolutionary patterns are dissimilar. Therefore, an applied estimation of the HCV epidemic history requires the proper selection of the rate priors, which should match the actual dataset so that they can fit for the subtype, the genomic region and even the length. By referencing the findings here, future evolutionary analysis of the HCV subtype 1a and 1b datasets may become more accurate and hence prove useful for tracing their patterns.     

Molecular Epidemiologic Characterization of a Clustering HCV Infection Caused by Inappropriate Medical Care in Heyuan City of Guangdong,China

Background

From November 2011 to January 2012, a number of clustering cases of HCV infection were reported in Zijin County, Heyuan City, Guangdong, China. Most patients in the clustering cases suspected that they could be infected due to inappropriate medical care in the clinic located at the Xiangshui road. However, the molecular epidemiology of the clustering cases remains unknown.

Methodology

The residents, living at Xiangshui Road, with HCV antibody positive reported from 2011 and 2012 were recruited. A survey of the HCV infected individuals from the clustering cases was conducted. Each participant underwent a questionnaire defining demographic characteristics and health care history. HCV serological test and viral load test were performed to confirm the infection status. Molecular phylogenetic analysis and Bayesian coalescence analysis were conducted to further confirm the HCV subtype distribution and to reconstruct the associated demographic history and time-scaled phylogeny among the clustering cases.

Principal Findings

The molecular phylogenetic analysis revealed that only two HCV subtypes, 2a and 6a, were found among the clustering cases. There was no close HCV subtype evolutionary relation was observed among patients from the same family. The 6a cluster showed higher viral loads than the 2a cluster. In addition, the Bayesian skyline plot analysis showed that both the HCV 2a and 6a subtype infections among the Heyuan cases experienced an “expansion-diminishment-expansion” featured dissemination. The 2a clustering infection occurred in 2004, and the 6a clustering cases originated in 2006.

Conclusions

The molecular epidemiological characters imply that the inappropriate medical practices were possibly associated with the clustering HCV cases in Heyuan City during 2011, 2012. Latent HCV subtypes 2a and 6a infection may cause the prevalence and become a new public health issue in Guangdong, China.     

The Molecular Epidemiological Study of HCV Subtypes among Intravenous Drug Users and Non-Injection Drug Users in China

BackgroundMore than half of intravenous drug users (IDUs) in China suffer from the Hepatitis C virus (HCV). The virus is also more prevalent in non-injection drug users (NIDUs) than in the general population. However, not much is known about HCV subtype distribution in these populations.MethodsOur research team conducted a cross-sectional study in four provinces in China. We sampled 825 IDUs and 244 NIDUs (1162 total), genotyped each DU’s virus, and performed a phylogenetic analysis to differentiate HCV subtypes.ResultsNucleic acid testing (NAT) determined that 82% percent (952/1162) of samples were HCV positive; we subtyped 90% (859/952) of these. We found multiple HCV subtypes: 3b (249, 29.0%), 3a (225, 26.2%), 6a (156, 18.2%), 1b (137, 15.9%), 6n (50, 5.9%), 1a (27, 3.1%), and 2a (15, 1.7%). An analysis of subtype distributions adjusted for province found statistically significant differences between HCV subtypes in IDUs and NIDUs.DiscussionHCV subtypes 3b, 3a, 6a, and 1b were the most common in our study, together accounting for 89% of infections. The subtype distribution differences we found between IDUs and NIDUs suggested that sharing syringes was not the most likely pathway for HCV transmission in NIDUs. However, further studies are needed to elucidate how NIDUs were infected.     

Genetic history of hepatitis C virus in Venezuela: high diversity and long time of evolution of HCV genotype 2

Background

The subtype diversity of the hepatitis C virus (HCV) genotypes is unknown in Venezuela.

Methodology/Principal Findings

Partial sequencing of the NS5B region was performed in 310 isolates circulating in patients from 1995 to 2007. In the samples collected between 2005 and 2007, HCV genotype 1 (G1) was the most common genotype (63%), composed as expected of mainly G1a and G1b. G2 was the second most common genotype (33%), being G2a almost absent and G2j the most frequent subtype. Sequence analysis of the core region confirmed the subtype assignment performed within the NS5b region in 63 isolates. The complete genome sequence of G2j was obtained. G2j has been described in France, Canada and Burkina Fasso, but it was not found in Martinique, where several subtypes of G2 circulate in the general population. Bayesian coalescence analysis indicated a most recent common ancestor (MRCA) of G2j around 1785, before the introduction of G1b (1869) and G1a (1922). While HCV G1a and G1b experienced a growth reduction since 1990, coincident with the time when blood testing was implemented in Venezuela, HCV G2j did not seem to reach growth equilibrium during this period.

Conclusions/Significance

Assuming the introduction of G2j from Africa during the slave trade, the high frequency of G2j found in Venezuela could suggest: 1- the introduction of African ethnic groups different from the ones introduced to Martinique or 2- the occurrence of a founder effect. This study represents an in-depth analysis of the subtype diversity of HCV in Venezuela, which is still unexplored in the Americas and deserves further studies.     

Similar aerosol emission rates and viral loads in upper respiratory tracts for COVID-19 patients with Delta and Omicron variant infection

Highlights
1 Aerosol emission rates of Delta or Omicron patients were similar.
2 Viral loads in upper respiratory tract of Alpha, Delta and Omicron patients were similar.
3 Viral loads in upper respiratory tract of vaccinated or unvaccinated Delta patients had no difference.     

Severe acute hepatitis in children with unknown aetiology,etiology analysis and the next action

Highlights
1. MIS-C and severe acute hepatitis might share the common pathogenic mechanism;
2. SARS-CoV-2 persistence throughout multiorgan and tissues;
3. Relationship between COVID-19 vaccines and severe acute hepatitis worth investigating.     

Genetic characterization and pathogenicity of a reassortant Eurasian avian-like H1N1 swine influenza virus containing an internal gene cassette from 2009 pandemic H1N1 virus

Highlights
1. Identification of a reassortant EA H1N1 SIV (SD/18) which isolated from a pig farm in Shandong, north China.
2. Phylogenetic analysis showed that SD/18 virus containing a complete internal gene cassette from pdm/09 virus.
3. The results of pathogenicity in mice showed that the mortality rate of SD/18 virus in mice could reach 100%.
4. The potential risk of EA lineage SIVs to humans is very high and we need to pay enough attention to the different reassortant EA H1N1 viruses.     

Identification of a novel hepacivirus in Mongolian gerbil (Meriones unguiculatus) from Shaanxi,China

Highlights:
1. The first hepacivirus detected in Mongolian gerbils from a plague zones in China.
2. A novel hepacivirus closely related to hepacivirus E and F.
3. Mongolian gerbils could be a potential animal model for hepacivirus pathogenicity.
4. Extending the genetic diversity and host range of hepaciviruses.     

New discovery of high-affinity SARS-CoV-2 spike S2 protein binding peptide selected by PhIP-Seq

Highlights
1 A peptide Spep-1 targeting S2 of SARS-CoV-2 spike protein was selected by PhIP-Seq.
2 Spep-1 showed nanomolar affinity and high specificity to spike protein.
3 S-1 based immunoassay can detect femtomolar spike antigen in spiked serum samples.
4 Spep-1 can be used in future on S2 recognition, virus tracing and drug delivery.     

Development of functional antibodies against influenza B virus by activation-induced cytidine deaminase in hybridoma cells

Highlights
1. Class-switch recombination was mimicked in hybridomas through a controllable expression system of activation-induced cytidine deaminase.
2. IgG antibodies were generated through this system in an anti-Flu B IgM hybridoma 7G1.
3. IgG1 and IgG2a subtypes of 7G1 present improved antiviral activity in vitro and in vivo.