Diverse Head-to-Tail Sequences in the Circular Genome of Human Bocavirus Genotype 1 among Children with Acute Respiratory Infections Implied the Switch of Template Chain in the Rolling-Circle Replication Model.

Head-to-tail sequences have been reported in human bocavirus (HBoV) 1-4. To reveal their features and functions, HBoV DNA was screened among respiratory specimens from pediatric patients with an acute respiratory infection (ARI) between April 2020 and December 2022, followed by HBoV genotyping. Head-to-tail sequences were detected using nested PCR, TA cloning, and Sanger sequencing, and these findings were confirmed by mNGS and amplicon sequencing. The secondary structure was predicted using the Mfold web server. The results indicated that head-to-tail sequences were detected in 42 specimens through TA cloning from 351 specimens positive for HBoV1 DNA, yielding 92 sequences into 32 types and 2 categories. Additionally, head-to-tail sequences were detected in 16 specimens by amplicon sequencing, yielding 60 sequences categorized into 23 types. The 374nt type, detected in 13 specimens, contains variants 374a and 374b, which differ in the unpaired loop regions of the palindrome or complementary reverse sequences, implying a switch of template chains during the replication process. The mNGS results in three specimens confirmed the presence of circular genome in copies below 1%. In conclusion, head-to-tail sequences of HBoV1 were common in children with ARI and were highly diverse in length and sequences. The variants may be generated by the switch of the template chain in the rolling-circle replication model.

Generation of rescued Japanese encephalitis virus genotype 1 from infectious full-size clone using reverse genetics.

Japanese encephalitis virus (JEV) is a pathogen responsible for high mortality and morbidity rates among children with encephalitis. Since JEV genotype 1 (GI) is the most prevalent strain in South Korea these days, corresponding research and vaccine development is urgently required. Molecular genetic studies on JEV vaccines can be boosted by obtaining genetically stable full-length infectious JEV complementary DNA (cDNA) clones. Furthermore, the significance of the reverse genetics system in facilitating molecular biological analyses of JEV properties has been demonstrated. This study constructed a recombinant JEV-GI strain using a reverse genetics system based on a Korean wild-type GI isolate (K05GS). RNA extracted from JEV-GI was used to synthesize cDNA, a recombinant full-length JEV clone, pTRE-JEVGI, was generated from the DNA fragment, and the virus was rescued. We performed in vitro and in vivo experiments to analyze the rescued JEV-GI virus. The rescued JEV-GI exhibited similar characteristics to wild-type JEV. These results suggest that our reverse genetics system can generate full-length infectious clones that can be used to analyze molecular biological factors that influence viral properties and immunogenicity. Additionally, it may be useful as a heterologous gene expression vector and help develop new strains for JEV vaccines.

Colon-derived Caco-2 cells support replication of hepatitis E virus genotype 1 strain Sar55 generated by reverse genetics.

The hepatitis E virus (HEV) is infecting over 20 million people annually with a high morbidity especially in pregnant women and immune-suppressed individuals. While HEV genotype 1 (HEV-1) infects only humans, genotype 3 (HEV-3) is zoonotic and commonly transmitted from infected animals to humans. Whereas a few reverse genetics systems enabling targeted genome manipulations exist for HEV-3, those for HEV-1 are still very limited, mainly because of inefficient cell culture replication. Here, the generation of HEV-1 strain Sar55 and HEV-3 strain 47832mc by transfecting in vitro-transcribed and capped virus genomes into different cell lines was attempted. Culture supernatants of colon-derived colorectal adenocarcinoma cell line Caco-2 contained HEV-1 and HEV-3 capable of infecting Caco-2 cells. Density gradient centrifugation analyses of culture supernatants confirmed that HEV-1 particles were quasi-enveloped in analogy to HEV-3 and that non-virion-associated capsid protein was secreted from cells. Following transfection or infection of Caco-2 cells, HEV-1 consistently reached higher titers than HEV-3 in culture supernatants, but HEV-1 generated by transfection of Caco-2 cells was unable to efficiently infect hepatoma cell lines PLC/PRF/5 or HuH7-Lunet BLR. Taken together, our results indicate that HEV-1 is able to exert a complete replication cycle in Caco-2 cells. An efficient cell culture system for this genotype will be useful for studying species tropism, but further research is required to determine the significance of HEV-1 replication in colon-derived cells.

Subclinical hepatitis E virus genotype 1 infection: The concept of "dynamic human reservoir".

Hepatitis E virus (HEV) is hyperendemic in South Asia and Africa accounting for half of total Global HEV burden. There are eight genotypes of HEV. Among them, the four common ones known to infect humans, genotypes 1 and 2 are prevalent in the developing world and genotypes 3 and 4 are causing challenge in the industrialized world. Asymptomatic HEV viremia in the general population, especially among blood donors, has been reported in the literature worldwide. The clinical implications related to this asymptomatic viremia are unclear and need further exploration. Detection of viremia due to HEV genotype 1 infection, apparently among healthy blood donors is also reported without much knowledge about its infection rate. Similarly, while HEV genotype 3 is known to be transmitted via blood transfusion in humans and has been subjected to screening in many European nations, instances of transmission have also been documented albeit without significant clinical consequences. Epidemiology of HEV genotype 1 in endemic areas often show waxing and waning pattern. Occasional sporadic occurrence of HEV infection interrupted by outbreaks have been frequently seen. In absence of known animal reservoir, where HEV exists in between outbreak is a mystery that needs further exploration. However, occurrence of asymptomatic HEV viremia due to HEV genotype 1 during epidemiologically quiescent period may explain that this phenomenon may act as a dynamic reservoir. Since HEV genotype 1 infection cannot cause chronicity, subclinical transient infection and transmission of virus might be the reason it sustains in interepidemic period. This might be the similar phenomenon with SARS COVID-19 corona virus infection which is circulating worldwide in distinct phases with peaks and plateaus despite vaccination against it. In view of existing evidence, we propose the concept of "Dynamic Human Reservoir." Quiescent subclinical infection of HEV without any clinical consequences and subsequent transmission may contribute to the existence of the virus in a community. The potential for transmitting HEV infection by asymptomatic HEV infected individuals by fecal shedding of virus has not been reported in literature. This missing link may be a key to Pandora's box in understanding epidemiology of HEV infection in genotype 1 predominant region.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

An Immunocompetent Mongolian Gerbil Model for Hepatitis E Virus Genotype 1 Infection.

BACKGROUND & AIMS: Hepatitis E virus (HEV), primarily genotype 1 (HEV-1), causes approximately 20.1 million infections, 44,000 deaths, and 3000 stillbirths annually. Current evidence indicates that HEV-1 is only transmitted in humans. Here, we evaluated whether Mongolian gerbils can serve as animal models for HEV-1 infection. METHODS: Mongolian gerbils were used for HEV-1 and hepatitis E virus genotype 3 infection experiments. HEV infection parameters, including detection of HEV RNA and HEV antigen, liver function assessment, and histopathology, were evaluated. RESULTS: We adapted a clinical isolate of HEV-1 for Mongolian gerbils by serial passaging in feces of aged male gerbils. The gerbil-adapted strain obtained at passage 3 induced a robust, acute HEV infection, characterized by stable fecal virus shedding, elevated liver enzymes, histopathologic changes in the liver, and seroconversion to anti-HEV. An infectious complementary DNA clone of the adapted virus was generated. HEV-1-infected pregnant gerbils showed a high rate of maternal mortality and vertical transmission. HEV RNA or antigens were detected in the liver, kidney, intestine, placenta, testis, and fetus liver. Liver and placental transcriptomic analyses indicated activation of host immunity. Tacrolimus prolonged HEV-1 infection, whereas ribavirin cleared infection. The protective efficacy of a licensed HEV vaccine was validated using this model. CONCLUSIONS: HEV-1 efficiently infected Mongolian gerbils. This HEV-1 infection model will be valuable for investigating hepatitis E immunopathogenesis and evaluating vaccines and antivirals against HEV.

Catalytic hairpin assembly triggering amplified DNAzyme-feedback for sensitive detection of hepatitis C virus genotype 1b.

Developing a simple, reliable, and sensitive hepatitis C virus (HCV) genetic sensing platform is of great significance for diagnosing diseases and selecting appropriate antiviral treatments. Herein, a tandem nucleic acid amplification strategy for sensitive detection of HCV genotype 1b (HCV-1b) was developed by stringing the catalytic hairpin assembly (CHA) and the triggered DNAzyme amplifier. The hairpin reactants were initiated by the target to produce lots of triggering double-stranded DNA sequences which can efficiently activate the subsequent blocked DNAzyme. Thereby, the continuous cleavage of substrate was realized, resulting in the fluorescence signal amplification. The DNA-based isothermal CHA-DNAzyme (CDz) sensing platform was successfully applied for sensitive detection of HCV-1b with the limit of detection (84 pM) and showed good selectivity. Moreover, the practical detection of target DNA in the complex biologic matrix indicated that the developing strategy had good potential for early HCV infection diagnosis.

Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan.

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.

First Report on Abnormal Renal Function in Acute Hepatitis E Genotype 1 Infection.

Impaired renal functions have been reported with Hepatitis E virus (HEV) infections, especially with genotypes 3 and 4. These complications were reported during the acute and chronic phases of infection. HEV genotype 1 causes acute infection, and the effect of HEV-1 infections on renal functions is not known. We examined the kidney function parameters in the serum of HEV-1 patients (AHE, n = 31) during the acute phase of infection. All of the included patients developed an acute self-limiting course of infection, without progression to fulminant hepatic failure. We compared the demographic, laboratory, and clinical data between AHE patients with normal kidney function parameters and those with abnormal renal parameters. Out of 31 AHE patients, 5 (16%) had abnormal kidney function tests (KFTs) during the acute phase of infection. Three patients had abnormal serum urea and creatinine, and two patients had either abnormal urea or creatinine. Four out of five patients had an estimated glomerular filtration rate (eGFR) below 60 mL/min/1.73 m2. AHE patients with abnormal KFTs were older and had a lower level of albumin, but a slightly elevated alanine transaminase (ALT) compared to AHE patients with normal KFTs. There were no significant differences between the two groups in terms of age, sex, liver transaminase levels, and the viral load. Similarly, the clinical presentations were comparable in both groups. Interestingly, these KFTs in patients with abnormal renal parameters returned to normal levels at the recovery. The serum creatinine level was not correlated with patients' age or liver transaminase levels, but it was significantly negatively correlated with albumin level. In conclusion, this study is the first report that evaluated KFTs in patients during the acute phase of HEV-1 infections. Impaired KFTs in some AHE patients resolved at convalescence. KFTs and renal complications should be monitored during HEV-1 infections.

Development and Characterization of Efficient Cell Culture Systems for Genotype 1 Hepatitis E Virus and Its Infectious cDNA Clone.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

SID: a new carbohydrate blood group system based on a well-characterized but still mysterious antigen of great pathophysiologic interest.

The high-prevalence blood group antigen, Sda, had been puzzling blood bankers and transfusionists for at least a decade when it was reported in 1967. The characteristic mix of agglutinates and free red blood cells (RBCs), caused by anti-Sda, is seen with the RBCs from 90 percent of individuals of European descent. However, only 2-4 percent of individuals are truly Sd(a-) and may produce anti-Sda. The antibodies, generally considered insignificant, may cause hemolytic transfusion reactions with high-expressing Sd(a+) RBCs (e.g., the unusual Cad phenotype, which can also be polyagglutinable). The Sda glycan, GalNAcß1-4(NeuAcα2-3)Gal-R, is produced in the gastrointestinal and urinary systems, while its origin on RBCs is more controversial. According to current theory, Sda is likely to be passively adsorbed in low amounts, except in Cad individuals, where it has been found on erythroid proteins and at higher levels. The long-standing hypothesis that B4GALNT2 encodes the Sda synthase was confirmed in 2019, since homozygosity for a variant allele with rs7224888:C produces a non-functional enzyme associated with most cases of the Sd(a-) phenotype. Thereby, the SID blood group system was acknowledged as number 038 by the International Society of Blood Transfusion. Although the genetic background of Sd(a-) was settled, questions remain. The genetic background of the Cad phenotype has not yet been determined, and the source of the RBC-carried Sda is unknown. Furthermore, the interest of Sda stretches beyond transfusion medicine. Some tantalizing examples are lowered antigen levels in malignant tissue compared with normal tissue and interference with infectious agents like Escherichia coli, influenza virus, and malaria parasites.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus.

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

High real-world effectiveness of 12-week elbasvir/grazoprevir without resistance testing in the treatment of patients with HCV genotype 1a infection in Norway.

BACKGROUND AND AIMS: The recommended treatment duration of hepatitis C virus (HCV) genotype 1a (GT1a) infection with elbasvir/grazoprevir (EBR/GZR) in the presence of a high baseline viral load and resistance associated substitutions (RAS) is 16 weeks with ribavirin added. The objective of this study was to evaluate the real-world effectiveness of 12 weeks of EBR/GZR without ribavirin and regardless of baseline viral load and RAS testing. METHOD: This retrospective, observational cohort study was performed at five Norwegian hospitals that did not systematically utilize RAS testing. All adult patients with chronic HCV GT1a and compensated liver disease who had received 12 weeks of EBR/GZR without ribavirin and baseline RAS testing, were included. The primary endpoint was sustained virologic response at week 12 (SVR12), or if not available, at week 4 (SVR4). RESULTS: We included 433 patients and attained SVR data on 388. The mean age was 45.7 years (22-73 years). 67.2% were male. HIV co-infection was present in 3.8% (16/424) and cirrhosis in 4% (17/424). The viral load was >800 000 IU/mL in 55.0% (235/427) of patients. Overall SVR was achieved in 97.2% (377/388). SVR was achieved in 98.3% (169/172) of those with viral load ≤800 000 IU/mL and in 96.2% (202/210) of those with viral load >800 000 IU/mL. CONCLUSION: We observed high SVR rates among patients with HCV GT1a infection treated with EBR/GZR for 12 weeks without ribavirin, with no regard to baseline viral load and no RAS testing.

Best therapy for the easiest to treat hepatitis C virus genotype 1b-infected patients.

BACKGROUND: The revolution in treatment of patients with chronic hepatitis C virus (HCV) infection dates back to the introduction of direct-acting antivirals (DAAs). The increase in efficacy was most pronounced in patients infected with genotype (GT) 1b, as this was the most poorly responsive population to treatment during the interferon era. AIM: To identify the most effective interferon-free therapy for GT1b-infected patients and to determine positive and negative predictors of virological response. METHODS: This real-world retrospective analysis included patients chronically infected with GT1b HCV whose data were obtained from the multicenter observational EpiTer-2 database. Treatment effectiveness was evaluated for each therapeutic regimen as the percentage of sustained virological responses (SVR). Assessment of the safety was based on the evaluation of the course of therapy, the occurrence of adverse events including serious ones, deaths during treatment and in the post 12-wk follow-up period. RESULTS: The studied population consisted of 11385 patients with a mean age of 53 ± 14.8 years and a female predominance (53.4%). The majority of them were treatment-naïve (74.6%) and patients with cirrhosis accounted for 24.3%. Of the DAA regimens used, 76.9% were GT-specific with ombitasvir/paritaprevir/ritonavir + dasabuvir ± ribavirin being the most used option (32.4%). A total of 10903 patients responded to treatment resulting in a 98.1% in the per-protocol analysis after excluding 273 patients without SVR data. The effectiveness of all regimens exceeded 90% and the highest SVR of 98.9% was achieved in patients treated with a combination of glecaprevir/pibrentasvir. Logistic regression analyses showed that the virologic response was independently associated with female sex [odds ratio (OR) = 1.67], absence of decompensated cirrhosis at baseline (OR = 2.42) and higher baseline platelets (OR = 1.004 per 1000/µL increase), while the presence of human immunodeficiency virus (HIV) coinfection significantly decreased the odds of response (OR = 0.39). About 95%-100% of patients completed therapy irrespective of the drug regimen. At least one adverse effect occurred in 10.9%-36.3% and most of them were mild. No treatment related deaths have been reported. CONCLUSION: We documented very high effectiveness and a good safety profile across all DAA regimens. Positive predictors of SVR were female sex, absence of decompensated cirrhosis at baseline and higher platelet count while HIV coinfection reduced the effectiveness.

Efficacy of 8- versus 12-week treatment with ledipasvir/sofosbuvir in chronic hepatitis C patients eligible for 8-week regimen in a real-world setting.

Introduction: Non-cirrhotic treatment-naive hepatitis C patients infected with genotype 1 can be treated with ledipasvir/sofosbuvir (LDV/SOF) for 8 weeks, but in practice this regimen is frequently extended up to 12 weeks at least in part due to insufficient real-world data supporting shortening of treatment. The aim of our study was to compare 8- and 12-week regimens' efficacy in patients eligible for 8-week therapy in a real-world setting. Material and methods: Data of HCV genotype 1 infected patients treated with LDV/SOF between 2015 and 2018 included in the EpiTer-2 database were analyzed with respect to patients' characteristics and length of treatment. Results: Among a total of 1718 patients treated with LDV/SOF, 679 were included in the analysis, 238 (35%) received 8-week regimen, whereas 441 were treated for 12 weeks although they fulfilled the criteria for a shorter course. The majority of patients were infected with genotype 1b (89%) and demonstrated minimal fibrosis (55%). The 12-week regimen was assigned significantly more frequently to patients with comorbidities, concomitant medications and advanced liver fibrosis. The sustained virologic response rate was similar after 8 (98%) and 12 (97%) weeks of therapy according to intent-to-treat analysis and reached 99% in both groups after exclusion of patients lost to follow-up. Conclusions: We confirmed high effectiveness regardless of treatment duration with LDV/SOF in non-cirrhotics infected with HCV genotype 1 eligible for the 8-week regimen according to the current label. This real-world study also demonstrated no need for addition of ribavirin (RBV) in this population and showed that shortening of treatment significantly improves the safety profile of LDV/SOF medication.

A small animal model of chronic hepatitis E infection using immunocompromised rats.

Background & Aims: HEV variants such as swine genotypes within Paslahepevirus species balayani (HEV-A) and rat HEV (Rocahepevirus ratti; HEV-C1) cause chronic hepatitis E in immunocompromised individuals. There are few reliable and accessible small animal models that accurately reflect chronic HEV infection. We aimed to develop an immunocompromised rat model of chronic hepatitis E infection. Methods: In this animal model infection study, rats were immunosuppressed with a drug combination (prednisolone, tacrolimus, and mycophenolate mofetil) commonly taken by transplant recipients. Rats were challenged with human- and rat-derived HEV-C1 strains or a human-derived HEV-A strain. Viral load, liver function, liver histology, humoural, and cellular immune responses were monitored. Results: A high-dose (HD) immunosuppressive regimen consistently prolonged human- and rat-derived HEV-C1 infection in rats (up to 12 weeks post infection) compared with transient infections in low-dose (LD) immunosuppressant-treated and immunocompetent (IC) rats. Mean HEV-C1 viral loads in stool, serum, and liver tissue were higher in HD regimen-treated rats than in LD or IC rats (p <0.05). Alanine aminotransferase elevation was observed in chronically infected rats, which was consistent with histological hepatitis and HEV-C1 antigen expression in liver tissue. None (0/6) of the HD regimen-treated, 5/6 LD regimen-treated, and 6/6 IC rats developed antibodies to HEV-C1 in species-specific immunoblots. Reversal of immunosuppression was associated with clearance of viraemia and restoration of HEV-C1-specific humoural and cellular immune responses in HD regimen-treated rats, mimicking patterns in treated patients with chronic hepatitis E. Viral load suppression was observed with i.p. ribavirin treatment. HD regimen-treated rats remained unsusceptible to HEV-A infection. Conclusions: We developed a scalable immunosuppressed rat model of chronic hepatitis E that closely mimics this infection phenotype in transplant recipients. Lay summary: Convenient small animal models are required for the study of chronic hepatitis E in humans. We developed an animal model of chronic hepatitis E by suppressing immune responses of rats with drugs commonly taken by humans as organ transplant rejection prophylaxis. This model closely mimicked features of chronic hepatitis E in humans.

Two mutations in the ORF1 of genotype 1 hepatitis E virus enhance virus replication and may associate with fulminant hepatic failure.

Hepatitis E virus (HEV) infection in pregnant women has a high incidence of developing fulminant hepatic failure (FHF) with significant mortality. Multiple amino acid changes in genotype 1 HEV (HEV-1) are reportedly linked to FHF clinical cases, but experimental confirmation of the roles of these changes in FHF is lacking. By utilizing the HEV-1 indicator replicon and infectious clone, we generated 11 HEV-1 single mutants, each with an individual mutation, and investigated the effect of these mutations on HEV replication and infection in human liver cells. We demonstrated that most of the mutations actually impaired HEV-1 replication efficiency compared with the wild type (WT), likely due to altered physicochemical properties and structural conformations. However, two mutations, A317T and V1120I, significantly increased HEV-1 replication. Notably, these two mutations simultaneously occurred in 100% of 21 HEV-1 variants from patients with FHF in Bangladesh. We further created an HEV-1 A317T/V1120I double mutant and found that it greatly enhanced HEV replication, which may explain the rapid viral replication and severe disease. Furthermore, we tested the effect of these FHF-associated mutations on genotype 3 HEV (HEV-3) replication and found that all the mutants had a reduced level of replication ability and infectivity, which is not unexpected due to distinct infection patterns between HEV-1 and HEV-3. Additionally, we demonstrated that these FHF-associated mutations do not appear to alter their sensitivity to ribavirin (RBV), suggesting that ribavirin remains a viable option for antiviral therapy for patients with FHF. The results have important implications for understanding the mechanism of HEV-1-associated FHF.

Isolation and Genome Sequencing of Hepatitis E Virus Genotype 1 Imported from India to Japan.

Hepatitis E virus (HEV) is the causative agent of viral hepatitis E. In Japan, HEV genotype 3 (G3) and G4 are predominantly detected, while G1, mainly imported from countries in continental Asia, is rare. In the present study, we detected a G1 HEV strain in a patient who visited Japan from India. When PLC/PRF/5 cells (subclone 4-21) were inoculated with a stool suspension from this patient, accumulation of HEV RNA was observed in the spent culture medium, indicating that HEV had been successfully isolated from this specimen. A nearly complete HEV genome was obtained by RT-PCR amplification. Phylogenetic analyses revealed that the newly isolated HEV strain, designated 9HE36c, belongs to subtype 1g of HEV G1.

Identification of a novel replication-competent hepatitis C virus variant that confers the sofosbuvir resistance.

Despite the excellent antiviral potency of direct-acting antivirals (DAAs) against hepatitis C virus (HCV), emergence of drug-resistant viral mutations remains a potential challenge. Sofobuvir (SOF), a nucleotide analog targeting HCV NS5B - RNA-dependent RNA polymerase (RdRp), constitutes a key component of many anti-HCV cocktail regimens and confers a high barrier for developing drug resistance. The serine to threonine mutation at the amino acid position 282 of NS5B (S282T) is the mostly documented SOF resistance-associated substitution (RAS), but severely hampers the virus fitness. In this study, we first developed new genotype 1b (GT1b) subgenomic replicon cells, denoted PR52D4 and PR52D9, directly from a GT1b clinical isolate. Next, we obtained SOF-resistant and replication-competent PR52D4 replicon by culturing the replicon cells in the presence of SOF. Sequencing analysis showed that the selected replicon harbored two mutations K74R and S282T in NS5B. Reverse genetics analysis showed that while PR52D4 consisting of either single mutation K74R or S282T could not replicate efficiently, the engineering of the both mutations led to a replication-competent and SOF-resistant PR52D4 replicon. Furthermore, we showed that the K74R mutation could also rescue the replication deficiency of the S282T mutation in Con1, another GT1b replicon as well as in JFH1, a GT2a replicon. Structural modeling analysis suggested that K74R might help maintain an active catalytic conformation of S282T by engaging with Y296. In conclusion, we identified the combination of two NS5B mutations S282T and K74R as a novel RAS that confers a substantial resistance to SOF while retains the HCV replication capacity.

Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis

ABSTRACT The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.