Cellular Release of Infectious Hepatitis C Virus Particles via Endosomal Pathways.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.

A household survey of intrafamily norovirus transmission.

Norovirus (NoV) is a leading cause of epidemic and sporadic gastroenteritis in people of all ages. Humans are the primary source of NoV and household contact is one of the risk factors for NoV transmission. However, the mechanisms underlying person-to-person NoV transmission are poorly understood. Here we conducted a survey to profile the frequency and characteristics of intrafamily NoV transmission. Stool samples were collected every week from three households between 2016 and 2020; the total number of samples was 1105. The detection of NoV and the genotyping were performed by reverse transcription-polymerase chain reaction targeting the capsid region and direct sequencing methods. NoV was detected in 3.4% of all samples. Eight NoV genotypes were identified. The most common genotype was GII.17, followed in order by GII.6, GI.6, GII.4, GI.3, and GI.2/GI.8/GI.9. Most NoV-positive samples were obtained from asymptomatic individuals. The highest number of NoV transmissions was found in household 3 (6 infections), followed by household 2 (2 infections), while household 1 had no NoV transmission, suggesting that asymptomatic NoV carriers play a major role in infection as NoV reservoirs in the households. Further clarification of the mode of infection will contribute to improved understanding and an appropriate prevention.

Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication.

IMPORTANCE: The Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.

Transcription Factor JunB Suppresses Hepatitis C Virus Replication.

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. Activation of JNK contributes to the development of liver diseases, including metabolic disorders, steatosis, liver cirrhosis and hepatocellular carcinoma. JNK is known to have numerous target genes, including JunB, a member of activator protein-1 transcription factor family. However, the roles of JunB in the HCV life cycle and HCV-associated pathogenesis remain unclear. To clarify a physiological role of JunB in HCV infection, we investigated the phosphorylation of JunB in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of JunB. The small interfering RNA (siRNA) knockdown of JunB significantly increased the amount of intracellular HCV RNA as well as the intracellular and extracellular HCV infectivity titers. Conversely, overexpression of JunB significantly reduced the amount of intracellular HCV RNA and the intracellular and extracellular HCV infectivity titers. These results suggest that JunB plays a role in inhibiting HCV propagation. Additionally, HCV-mediated JunB activation promoted hepcidin promoter activity and hepcidin mRNA levels, a key factor in modulating iron homeostasis, suggesting that JunB is involved in HCV-induced transcriptional upregulation of hepcidin. Taken together, we propose that the HCV-induced ROS/JNK/JunB signaling pathway plays roles in inhibiting HCV replication and contributing to HCV-mediated iron metabolism disorder.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Complete genome analyses of G12P[8] rotavirus strains from hospitalized children in Surabaya, Indonesia, 2017-2018.

Rotavirus A (RVA) is a major viral cause of acute gastroenteritis (AGE) worldwide. G12 RVA strains have emerged globally since 2007. There has been no report of the whole genome sequences of G12 RVAs in Indonesia. We performed the complete genome analysis by the next-generation sequencing of five G12 strains from hospitalized children with AGE in Surabaya from 2017 to 2018. All five G12 strains were Wa-like strains (G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) and were clustered into lineage-III of VP7 gene phylogenetic tree. STM430 sample was observed as a mixed-infection between G12 and G1 strains: G12/G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. A phylogenetic tree analysis revealed that all five Indonesian G12 strains (SOEP379, STM371, STM413, STM430, and STM433) were genetically close to each other in all 11 genome segments with 98.0%-100% nucleotide identities, except VP3 and NSP4 of STM430, suggesting that these strains have originated from a similar ancestral G12 RVA. The VP3 and NSP4 genome segments of STM430-G12P[8] were separated phylogenetically from those of the other four G12 strains, probably due to intra-genotype reassortment between the G12 and G1 Wa-like strains. The change from G12P[6] lineage-II in 2007 to G12P[8] lineage-III 2017-2018 suggests the evolution and diversity of G12 RVAs in Indonesia over the past approximately 10 years.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

Hepatitis C Virus-Induced ROS/JNK Signaling Pathway Activates the E3 Ubiquitin Ligase Itch to Promote the Release of HCV Particles via Polyubiquitylation of VPS4A.

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle remain unclear. We sought to identify a novel role of the ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The small interfering RNA (siRNA) knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in the release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase, VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Coimmunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that the HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles. IMPORTANCE The ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that the HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis.

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

NS5A-ISGylation via Lysine 26 Has a Critical Role for Efficient Propagation of Hepatitis C Virus Genotype 2a.

We previously reported that hepatitis C virus (HCV) NS5A (1b, Con1) protein accepts covalent ISG15 conjugation at specific lysine (Lys) residues (K44, K68, K166, K215 and K308), exhibiting proviral effects on HCV RNA replication. Here we investigated a role of NS5A-ISGylation via Lys residues in HCV propagation using HCV infectious clone. The alignment of amino acid sequences revealed that 5 Lys residues (K20, K26, K44, K139, and K166) of the 13 Lys residues within NS5A (genotype 2a, JFH1 strain) were conserved compared to those of HCV (genotype 1b, Con1 strain). The cell-based ISGylation assay revealed that the K26 residue in the amphipathic helix (AH) domain and the K139 residue in domain I of NS5A (2a, JFH1) had the potential to accept ISGylation. Use of the HCV replicon carrying luciferase gene revealed that the K26 residue but not K139 residue of NS5A (2a, JFH1) was important for HCV RNA replication. Furthermore, cell culture HCV revealed that the mutation with the K26 residue in combination with K139 or K166 on NS5A (2a, JFH1) resulted in complete abolishment of viral propagation, suggesting that the K26 residue collaborates with either the K139 residue or K166 residue for efficient HCV propagation. Taken together, these results suggest that HCV NS5A protein has the potential to accept ISGylation via specific Lys residues, involving efficient viral propagation in a genotype-specific manner.

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication.

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

Prevalence and Distribution of Rotavirus Genotypes Among Children With Acute Gastroenteritis in Areas Other Than Java Island, Indonesia, 2016-2018.

Group A rotaviruses (RVAs) are the leading cause of acute gastroenteritis, which is often associated with severe symptoms in children under 5 years old. Genetic reassortments and interspecies transmission commonly occur, resulting in a great diversity of RVA circulating in the world. The aim of this study is to determine the prevalence and distribution of RVA genotypes among children in Indonesia over the years 2016-2018 across representative areas of the country. Stool samples were collected from 202 pediatric patients with acute gastroenteritis in three regions of Indonesia (West Nusa Tenggara, South Sumatra, and West Papua) in 2016-2018. Rotavirus G and P genotypes were determined by reverse transcription PCR (RT-PCR) and direct sequencing analysis. The prevalences of RVA in South Sumatra (55.4%) and West Papua (54.0%) were significantly higher than that in East Java (31.7%) as determined in our previous study. The prevalence in West Nusa Tenggara (42.6%) was the lowest among three regions, but higher than that in East Java. Interestingly, equine-like G3 rotavirus strains were found as predominant strains in South Sumatra in 2016 and in West Papua in 2017-2018. Moreover, the equine-like G3 strains in South Sumatra detected in 2016 were completely replaced by human G1 and G2 in 2018. In conclusion, RVA infection in South Sumatra and West Papua was highly endemic. Equine-like G3 strains were also spread to South Sumatra (West Indonesia) and West Papua (East Indonesia), as well as Java Island. Dynamic change in rotavirus genotypes from equine-like G3 to human genotypes was also observed. Continuous monitoring may be warranted in isolated areas in Indonesia.

Molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in East Java, Indonesia in 2015-2019.

Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5 years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2 years old showed a significantly higher prevalence than those ≥2 years old (P = 0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.

ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called ISGylation. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, the ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A ISGylation in HCV replication is proviral or antiviral. Here, we investigated the role of NS5A ISGylation in HCV replication by using HCV RNA replicons that encode a mutation at each lysine (Lys) residue of the NS5A protein. Immunoblot analyses revealed that 5 Lys residues (K44, K68, K166, K215, and K308) of the 14 Lys residues within NS5A (genotype 1b, Con1) have the potential to accept ISGylation. We tested the NS5A ISGylation among different HCV genotypes and observed that the NS5A proteins of all of the HCV genotypes accept ISGylation at multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence derived from all of the HCV genotypes suggesting that NS5A ISGylation enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A ISGylation functions as a proviral factor and promotes HCV replication via the recruitment of CypA.IMPORTANCE Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here, we demonstrate that HCV NS5A protein accepts ISG15 conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A ISGylation. We obtained evidence suggesting that NS5A ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.

G2P[4] rotavirus outbreak in Belu, East Nusa Tenggara Province, Indonesia, 2018.

Rotavirus is a major cause of acute gastroenteritis (AGE) in children worldwide. However, rotavirus outbreak has rarely been reported in Indonesia. This study aims to identify the causative agent for AGE outbreak among children in Belu, East Nusa Tenggara, Indonesia in 2018. All the samples were negative for bacteria (Salmonella, V. cholera) and Norovirus. Ten out of 11 stool samples were rotavirus-positive by immunochromatography testing. Reverse-transcription polymerase chain reaction (RT-PCR) and phylogenetic analyses revealed that rotavirus G2P[4] was the possible causative agent for the AGE outbreak, although sample size was limited. These findings suggest that the AGE outbreak was caused by rotavirus G2P[4], highlighting the importance of rotavirus surveillance.

Predominance of norovirus GI.4 from children with acute gastroenteritis in Jambi, Indonesia, 2019.

Norovirus (NoV) is one of the most important viral causes of acute gastroenteritis (AGE) in children worldwide. Only a few studies have reported AGE with NoV-positive in some cities in Indonesia. This study aimed to investigate the incidence and clinical characteristic of NoV infection, and also genotype distribution of NoV in children with AGE in Jambi, as the capital and the largest city of Jambi province, Indonesia. Stool samples were collected from children (≤15 years of age) with AGE at three participating hospitals in Jambi from February to April 2019. The detection of NoV and its genotyping were carried out by reverse-transcriptase polymerase chain reaction and direct sequencing. Of the 91 stool samples collected, 14 (15.4%) were positive for NoV. Fever, vomiting, and severe diarrhea were commonly observed in AGE with NoV, while level of dehydration was statistically significant difference between children with NoV-positive and those with NoV-negative. The most prevalent genotype was GI.4 (42.9%), followed by GII.6 (28.6%) and some other genotypes. Interestingly, this study found the predominance of GI.4, differed from previous reports in Indonesia. Continuously investigation of the circulating genotype is needed to control the NoV-infected AGE.

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015-2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia's national immunization program.

Improvement of Rotavirus Genotyping Method by Using the Semi-Nested Multiplex-PCR With New Primer Set.

Rotavirus A (RVA) is a major cause of gastroenteritis in infants and young children. After vaccine introduction, RVA surveillance has become more important for monitoring changes in genotype distribution, and the semi-nested multiplex-PCR is a popular method for RVA genotyping. In particular, the VP7 primer set reported by Gouvea and colleagues in 1990 is still widely used worldwide as the recommended WHO primer set in regional and national reference RVA surveillance laboratories. However, this primer set yielded some mistakes with recent epidemic strains. The newly emerged equine-like G3 strains were mistyped as G1, G8 strains were mistyped as G3, the G9 lineage 3 strains showed very weak band, and the G9 lineage 6 strains showed a G9-specific band and a non-specific band. Gouvea's standard protocol has become relatively unreliable for identifying genotypes correctly. To overcome this limitation, we redesigned the primer set to include recent epidemic strains. Our new primer set enabled us to correctly identify the VP7 genotypes of representative epidemic strains by agarose gel electrophoresis (G1, G2, human typical G3, equine-like G3, G4, G8, G9, and G12). We believe that the multiplex-PCR method with our new primer set is a useful and valuable tool for surveillance of RVA epidemics.

Post-vaccinated asymptomatic rotavirus infections: A community profile study of children in Surabaya, Indonesia.

BACKGROUND: Rotavirus gastroenteritis accounts for significant childhood morbidity and mortality worldwide. Vaccination using RotarixTM (GSK) and RotaTeq® (Merck) was introduced due to the tremendous disease burden. The possibility of asymptomatic infections following vaccinations was poorly understood. This study examined rotavirus cases in post-vaccinated children, their clinical manifestations and the genotypes of isolated strains. METHODS: Stool samples of healthy, vaccinated children under 5 years of age in Surabaya were collected monthly for 1 year between January 2016 and February 2017. Episodes of gastroenteritis were reported, and samples were collected. Rotavirus was identified using multiplex reverse transcription Polymerase Chain Reaction (QIAGEN, Inc., Valencia, CA). Clinical manifestations were measured using the Vesikari score. The genotype was analyzed by Applied Biosystems (Foster, CA). RESULTS: A total of 109 stool samples were collected from 30 subjects, of which 22 received Rotarix; 8 RotaTeq. Nine out of 109 samples were collected during diarrhea episodes of 8 subjects. Two asymptomatic rotavirus infections were identified by RT-PCR. The genotypes isolated were G1P[8] and G3P[8]. CONCLUSIONS: Asymptomatic rotavirus infections can occur in post-vaccinated children. Strains identified were homologous to serotypes eliciting gastroenteritis in unvaccinated children of the same community.