Investigation of transfusion associated hepatitis C virus infection in Taiwan, 2015-2018.

Continuous strengthening of the safety of blood products to reduce the risk of transfusion-transmitted HCV in recipients is an important issue of Taiwanese government concern. Since 2013, highly sensitivity serology and NAT assays were simultaneously used for blood donation screening to shorten the window period of HIV, HBV and HCV infections. 15 cases of suspected transfusion-transmitted HCV infection were analyzed in 2015-2018. No HCV nucleic acid was detected among a total 91 bags of donated blood. Eleven cases among the 15 suspected recipients were positive for HCV nucleic acid, and 9 recipients had genotype results. Of these 9 recipients, five for genotype 1b (5/9, 55.6%), three for genotype 2a (3/9, 33.3%) and one for genotype 2b (1/9, 11.1%). We will continuously monitor the blood safety of recipients. There have been no confirmed cases of acute hepatitis C (AHC) infection due to transfusions of HCV contaminated blood product in 2015-2018 in Taiwan.

Foodborne hepatitis A outbreak associated with bakery products in northern Germany, 2012.

In October 2012, a hepatitis A (HA) outbreak with 83 laboratory-confirmed cases occurred in Lower Saxony. We defined primary outbreak cases as people with laboratory-confirmed HA and symptom onset between 8 October and 12 November 2012, residing in or visiting the affected districts. Secondary outbreak cases were persons with symptom onset after 12 November 2012 and close contact with primary cases. We identified 77 primary and six secondary cases. We enrolled 50 primary cases and 52 controls matched for age and sex, and found that 82% of cases and 60% of controls had consumed products from a particular bakery (OR=3.09; 95% CI: 1.15­8.68). Cases were more likely to have eaten sweet pastries (OR=5.74; 95% CI: 1.46­22.42). Viral isolates from five selected cases and three positively tested surfaces in the bakery had identical nucleotide sequences. One additional identical isolate derived from a salesperson of the bakery suffering from a chronic disease that required immunosuppressive treatment. Epidemiological and laboratory findings suggested that the salesperson contaminated products while packing and selling. Future risk assessment should determine whether food handlers with chronic diseases under immunosuppressive treatment could be more at risk of contaminating food and might benefit from HAV immunisation.

[Experience of application of multiplex qPCR for differential diagnostics of intestinal viral infections].

AIM: Evaluate the effectiveness of multiplex reverse transcription (RT) and polymerase chain reaction with fluorescence detection in real time mode (qPCR) methods for differential detection of 11 groups of intestine viruses (adenoviruses, enteroviruses, polioviruses, hepatitis A and E viruses, group A and C rotaviruses, orthoreoviruses, noroviruses, sapoviruses and astroviruses) in various biological samples. MATERIALS AND METHODS: Panels of virus isolates and clinical samples characterized by reference methods were used to evaluate sensitivity of detection of various intestine viruses. Nucleic acids were isolated from study samples and multiplex RT and qPCR were carried out. RESULTS: Sensitivity of laboratory reagent kit (LRK) when compared with results obtained from reference methods was 100% for rotavirus A, adenovirus, enterovirus and norovirus, 88.9% for hepatitis E virus and 92.3% for hepatitis A virus, and diagnostic specificity - 99.4%. During analysis of 697 clinical samples from patients with acute intestine infection symptoms nucleic acids of various intestine viruses were isolated in 71.7%. CONCLUSION: Multiplex qRT-PCR was shown as an effective method of etiologic diagnostics of an intestine viral infection. Use of LRK was demonstrated to establish etiology of intestine diseases in 63 - 72% and in children with watery diarrhea - in approximately 90% of cases.

Internal initiation of translation.

Although the 5' cap-dependent scanning mechanism can account for the translational initiation of most mRNAs in eukaryotic cells, several viral and cellular mRNAs contain nucleotide sequences in their 5' non-coding regions that can mediate binding of ribosomes to the mRNA, regardless of the modification state of the 5' ends. During the past year, some nuclear proteins normally involved in RNA processing have been shown also to facilitate 'internal' ribosome binding. Unexpected dual functions have, therefore, been suggested for these RNA-binding proteins, in both RNA biogenesis in the nucleus and RNA translation in the cytoplasm.

Identification of a conserved RNA replication element (cre) within the 3Dpol-coding sequence of hepatoviruses.

Internally located, cis-acting RNA replication elements (cre) have been identified within the genomes of viruses representing each of the major picornavirus genera (Enterovirus, Rhinovirus, Aphthovirus, and Cardiovirus) except Hepatovirus. Previous efforts to identify a stem-loop structure with cre function in hepatitis A virus (HAV), the type species of this genus, by phylogenetic analyses or thermodynamic predictions have not succeeded. However, a region of markedly suppressed synonymous codon variability was identified in alignments of HAV sequences near the 5' end of the 3D(pol)-coding sequence of HAV, consistent with noncoding constraints imposed by an underlying RNA secondary structure. Subsequent MFOLD predictions identified a 110-nucleotide (nt) complex stem-loop in this region with a typical AAACA/G cre motif in its top loop. A potentially homologous RNA structure was identified in this region of the avian encephalitis virus genome, despite little nucleotide sequence relatedness between it and HAV. Mutations that disrupted secondary RNA structure or the AAACA/G motif, without altering the amino acid sequence of 3D(pol), ablated replication of a subgenomic HAV replicon in transfected human hepatoma cells. Replication competence could be rescued by reinsertion of the native 110-nt stem-loop structure (but not an abbreviated 45-nt stem-loop) upstream of the HAV coding sequence in the replicon. These results suggest that this stem-loop is functionally similar to cre elements of other picornaviruses and likely involved in templating VPg uridylylation as in other picornaviruses, despite its significantly larger size and lower free folding energy.

Viral hepatitis among men who have sex with men, epidemiology and public health consequences.

Viral hepatitis causes major disease burden worldwide, due to the chronic hepatitis sequelae: cirrhosis and primary liver cancer. Transmission of viral hepatitis is a problem not only in low-income countries, but also in high-income ones where viral hepatitis is a frequently occurring infection among men who have sex with men (MSM). Although the transmission routes of the three main hepatitis viruses, A, B and C, differ, MSM mainly acquire viral hepatitis during sexual contact. Vaccination programmes (only available for hepatitis A and B), raising awareness, and screening can be used to prevent transmission. However, despite the introduction of such methods in many high-income countries, the spread of viral hepatitis among MSM is still ongoing. This paper provides an overview of sexually acquired hepatitis A, B, and C among MSM in high-income countries, using recent insights obtained through molecular epidemiology, with the aim to raise awareness, improve vaccination coverage, and stimulate prevention programs.

A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

[Duration of viremia and fecal shedding of the virus in hepatitis A infected children]. / Duración de la viremia y lla excreción fecal del virus en niños con hepatitis A.

Hepatitis A virus (HAV) infection is endemic in Argentina. Molecular tools have allowed HAV RNA detection to be extent to sera and feces from patients with different clinical backgrounds. We compare the sensitivity of the RT-PCR protocol we follow using primers targeting different genomic regions and VP3 C terminal was the most sensitive. Sequential sera and fecal samples were obtained from 20 children with acute self limited Hepatitis A. HAV RNA was detectable in 18/20 children if sera and stool specimens were collected at the onset of symptoms and in 19/20 if a later sample was considered. HAV RNA was detectable in serum from 9/20 patients until day 30 and in feces from 2 patients until day 60 and until day 90 in one. Genomic sequences from VP1/2A region in 8 samples showed they all belong to subgenotype IA although they were different between them. HAV RNA was detectable only in 1/11 sera from children with acute liver failure when VP3 C terminal fragment was searched and it belonged to genotype I. Universal vaccination in one year old children was recently implemented in Argentina and it will dramatically enable the decrease of the viral circulation, making new sources of infection emerge and allowing the introduction of new genotypes. The application of molecular tools to the study of the natural history of infection and to the epidemiologic surveillance may contribute to efficient control and lead to rational decisions in public health policies.

A novel hepatovirus identified in wild woodchuck Marmota himalayana.

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.

Site-directed mutagenesis of hepatitis A virus protein 3A: effects on membrane interaction.

Due to a stretch of hydrophobic amino acids, protein 3A of hepatitis A virus (HAV) has been suggested to act as a membrane anchor or a carrier of the genome-linked protein 3B (VPg) during viral RNA synthesis. Mutagenesis analysis was performed in order to elucidate the role of the N- and C-terminal tracts of protein 3A in cell membrane interaction. Expression of the mutated proteins in E. coli cells demonstrated that the presence of positively charged residues at the C-terminus is not required for membrane anchoring. Changes in the primary sequence involving charged amino acids at the N- and C-termini critically influenced the ability of the protein 3A of a cytopathic strain of HAV to change bacterial membrane permeability. This result demonstrates the strict correlation between the structure and pore-forming potential of HAV protein 3A.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Development of array-based technology for detection of HAV using gold-DNA probes.

A sensitive method for detection of Hepatitis A virus (HAV) by utilizing gold-DNA probe on an array was developed. Amino-modified oligodeoxynucleotides at the 5' position were arrayed on an activated glass surface to function as capture probes. Sandwich hybridization occurred among capture probes, the HAV amplicon, and gold nanoparticlesupported oligonucleotide probes. After a silver enhancement step, signals were detected by a standard flatbed scanner or just by naked eyes. As little as 100 fM of HAV amplicon could be detected on the array. Therefore, the array technology is an alternative to be applied in detection of HAV due to its low-cost and high-sensitivity.

Fulminant non--A-G viral hepatitis leading to liver transplantation.

BACKGROUND: All hepatotropic viruses are known to cause fulminant hepatic failure (FHF). However, in 30% to 40% of patients with FHF, the precise cause remains unknown. We aimed to better define this subgroup. METHODS: We evaluated the clinical course and outcome of 7 patients admitted during a 22-month period with fulminant viral hepatitis leading to liver transplantation; none had serologic or molecular evidence of hepatitis A, B, C, D, E, or G viral infection, thus the term non-A-G viral hepatitis. All known etiologies of FHF were excluded. RESULTS: All patients had prodromal symptoms suggestive of viral causes. Mean age was 30 years. The interval between onset of jaundice and appearance of encephalopathy was 23 days (range, 4-50 days). Five patients had grade III/IV encephalopathy. Serum alanine aminotransferase levels showed a single peak of activity. The duration between first symptoms and liver transplantation was 28 days (range, 12-71 days). Results of histological study of the explanted liver showed submassive (4 patients) or massive (3 patients) hepatocyte necrosis. In all patients, results of polymerase chain reaction analysis did not detect hepatitis B virus DNA, hepatitis C virus RNA, or hepatitis G virus RNA in the explanted liver. After transplantation, 2 patients showed (6 months later) increased liver enzyme levels of undetermined cause, and results of a liver biopsy showed mild lobular hepatitis; 1 patient had lymphoproliferative disorder (Epstein-Barr virus-originated); and 1 patient, aplastic anemia, which is known to be associated with seronegative viral hepatitis. The latter patient died, whereas the other 6 patients are alive (survival rate, 86%). CONCLUSIONS: Our patients with non-A-G viral hepatitis had a severe acute onset with progressive FHF requiring liver transplantation. There is some suggestion of recurrent viral disease after transplantation implicating other unknown viruses in the etiology.

Discovery of a Novel Hepatovirus (Phopivirus of Seals) Related to Human Hepatitis A Virus.

UNLABELLED: Describing the viral diversity of wildlife can provide interesting and useful insights into the natural history of established human pathogens. In this study, we describe a previously unknown picornavirus in harbor seals (tentatively named phopivirus) that is related to human hepatitis A virus (HAV). We show that phopivirus shares several genetic and phenotypic characteristics with HAV, including phylogenetic relatedness across the genome, a specific and seemingly quiescent tropism for hepatocytes, structural conservation in a key functional region of the type III internal ribosomal entry site (IRES), and a codon usage bias consistent with that of HAV. IMPORTANCE: Hepatitis A virus (HAV) is an important viral hepatitis in humans because of the substantial number of cases each year in regions with low socioeconomic status. The origin of HAV is unknown, and no nonprimate HAV-like viruses have been described. Here, we describe the discovery of an HAV-like virus in seals. This finding suggests that the diversity and evolutionary history of these viruses might be far greater than previously thought and may provide insight into the origin and pathogenicity of HAV.

The distance between the 3'-pyrimidine-rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA.

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5'-terminal extra-cistronic region of HAV RNA were inserted in the intergenic region of the bi-cistronic plasmid pSV-GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS-1 cells, the transient expression of both genes was quantified. The importance of the 3'-PRT appeared to be strongly influenced by the length of the 'spacer' sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the 'spacer' back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3'-PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5'-PRT fluctuated: while integrity of domain 100-106 was always stringently required for initiation to occur, the activity of domain 113-118 paralleled that of the 3'-PRT, and the opposite applied to domain 121-126, whose contribution became relevant only after switching off the 3'-PRT. Systematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.

Variations in genome fragments coding for RNA polymerase in human and simian hepatitis A viruses.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.

Relapsing hepatitis A. Review of 14 cases and literature survey.

We have reviewed our experience with 14 cases of relapsing hepatitis A (RH-A), as well as 68 cases reported in the literature. Relapse occurs in 3 to 20% of patients with acute hepatitis A, and rarely takes the form of a polyphasic disease (multiple relapses). After a stage of typical hepatitis A, remission phase ensues, with partial or complete resolution of clinical and biochemical manifestations. Relapse usually occurs after a short period (usually less than 3 weeks). Relapse is usually clinically milder than the first phase, with variable liver function abnormalities and a tendency toward more marked cholestatic features. Not uncommonly, immune manifestations occur during this phase, including purpura, nephritis, and arthralgia, with common laboratory findings of rheumatoid factor as well as false-positive reaction to HCV-EIA tests. The clinical course in relapsing hepatitis A is almost always benign, and uneventful recovery is the rule with few exceptions. Steroid treatment, first reported in the present series, resulted in marked clinical improvement. Preliminary results suggest that R-HA is associated with a continuing viremia as well as shedding of virus in stools during the relapse phase. The pathogenesis of R-HA probably involves an interaction between persistent viral infection and immune mechanisms responding to the continuing antigenic stimulation.

Competitor template RNA for detection and quantitation of hepatitis A virus by PCR.

PCR was used to introduce a 63-bp deletion into the putative RNA replicase coding sequence of hepatitis A virus. RNA was synthesized in vitro from the deletion mutant cloned into a transcription vector. Upon amplification by PCR, cDNA made from the competitor RNA generated an amplified fragment that could be easily distinguished from the product generated from wild-type hepatitis A virus genomic RNA by gel electrophoresis, when the same primers were used, without further manipulation. The competitor RNA was used as a positive control in PCR-based detection of very low copy numbers of hepatitis A virus genomic RNA in the presence of unrelated hard-shell clam RNA. When the competitor RNA was used for competitive PCR to quantitate wild-type RNA, the presence of one template at a 10-fold to 100-fold higher level almost completely inhibited product formation from the underrepresented template. The competitor RNA should be useful as a control for reverse transcription and PCRs to determine hepatitis A virus genome RNA when accidental contamination of test samples by a wild-type positive control template would compromise the results.