The European tiered approach for virucidal efficacy testing - rationale for rapidly selecting disinfectants against emerging and re-emerging viral diseases.

When facing an emerging virus outbreak such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a quick reaction time is key to control the spread. It takes time to develop antivirals and vaccines, and implement vaccination campaigns. Therefore, preventive measures such as rapid isolation of cases and identification and early quarantine of cases' close contacts-as well as masks, physical distancing, hand hygiene, surface disinfection and air control-are crucial to reduce the risk of transmission. In this context, disinfectants and antiseptics with proven efficacy against the outbreak virus should be used. However, biocidal formulations are quite complex and may include auxiliary substances such as surfactants or emollients in addition to active substances. In order to evaluate disinfectants' efficacy objectively, meaningful efficacy data are needed. Therefore, the European Committee for Standardisation technical committee 216 'Chemical disinfectants and antiseptics' Working Group 1 (medical area) has developed standards for efficacy testing. The European tiered approach grades the virucidal efficacy in three levels, with corresponding marker test viruses. In the case of SARS-CoV-2, disinfectants with proven activity against vaccinia virus, the marker virus for the European claim 'active against enveloped viruses', should be used to ensure effective hygiene procedures to control the pandemic.

The unique role of domain 2A of the hepatitis A virus precursor polypeptide P1-2A in viral morphogenesis.

The initial step during assembly of the hepatitis A virus particle is driven by domain 2A of P1-2A, which is the precursor of the structural proteins. The proteolytic removal of 2A from particulate VP1-2A by an as yet unknown host enzyme presumably terminates viral morphogenesis. Using a genetic approach, we show that a basic amino acid residue at the C-terminus of VP1 is required for efficient particle assembly and that host proteases trypsin and cathepsin L remove 2A from hepatitis A virus particles in vitro. Analyses of insertion mutants in the C-terminus of 2A reveal that this part of 2A is important for liberation of P1-2A from the polyprotein. The data provide the first evidence that the VP1/2A junction is involved in both viral particle assembly and maturation and, therefore, seems to coordinate the first and last steps in viral morphogenesis.

Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation.

Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C(pro)). However, PABP is cleaved by HAV 3C(pro) in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5' nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.

Immunogenic epitopes on the surface of the hepatitis A virus capsid: Impact of secondary structure and/or isoelectric point on chimeric virus assembly.

Hepatitis A virus (HAV) protein 2A has the capacity to harbor and expose a short foreign epitope. The chimeric virus, HAV-gp41, bearing seven amino acids of the 2F5 epitope of the HIV glycoprotein gp41, was shown to replicate in cell culture and laboratory animals and to induce a humoral immune response. As an extension of this work, we now investigated the possibility to insert longer epitopes, their impact on genetic stability, and the production of chimeric HAV. Twenty-seven amino acid residues of either HIV gp41, comprising the 2F5 epitope, or of a mimotope (F78) of the hypervariable region 1 of the hepatitis C virus (HCV) envelope protein E2 were inserted near the C-terminus of HAV 2A and viral capsid formation and replication were studied. The genome of the chimeric virus (HAV-F78) had reduced replication ability, yet the sedimentation profile of the chimeric particles was unchanged and the HCV sequence was maintained over serial viral passages. In contrast, no capsids were formed when an extended HIV epitope of 27 residues was inserted, precluding the rescue of infectious chimeric virus. Based on structural analyses, the data suggest that the isoelectric point (pI) and/or the secondary structure of the chimeric proteins are essential determinants that affect HAV particle formation for which protein 2A serves as an assembly signal.

Immunogenicity of a chimeric hepatitis A virus (HAV) carrying the HIV gp41 epitope 2F5.

Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.

An outbreak of hepatitis A virus infection with a high case-fatality rate among injecting drug users.

BACKGROUND/AIMS: In 2002, the first reported outbreak of hepatitis A virus (HAV) infection involving mostly intravenous drug users (IDU) occurred in Italy. We attempted a thorough evaluation of the outbreak, including epidemiological, clinical and virological analyses. METHODS: We conducted an epidemiological investigation, including a case-control study, to identify the source and the modes of HAV transmission. Hepatitis B and C (HCV) viruses and human immunodeficiency virus (HIV) coinfections were clinically analysed. Sequence analysis of the VP1/2A junction of the HAV isolates was also performed. RESULTS: Of the 47 symptomatic cases, 35 were IDUs. The only associated risk factor was contact (not related to injecting practices) with a jaundiced person (odds ratio: 5.8; 95% confidence interval: 1.3-29.9). Of the cases, 58% were anti-HCV positive and 4.7% anti-HIV positive. Three individuals died of acute liver failure: 2 were HCV-coinfected alcohol abusers, with underlying liver cirrhosis; 1 was HCV/HIV-coinfected. HAV-RNA was found in 15 of the 24 tested patients: genotype IB (8 cases) and IIIA (7 cases) were detected. CONCLUSIONS: HAV was probably transmitted through the fecal-oral route, although parenteral transmission cannot be excluded. The high fatality rate was probably due to severe underlying liver damage. The occurrence of this outbreak highlights the need for routine HAV vaccination for IDUs.

Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface.

Hepatitis A virus (HAV) protein 2A has been demonstrated to be involved in virus morphogenesis and suggested to be located on the surface of the particle. To determine whether this protein can function as a target structure to harbor and expose foreign epitopes on HAV particles, a full-length HAV cDNA, containing a seven amino acid stretch of human immunodeficiency virus type 1 (HIV-1) envelope protein gp41, was constructed. Following vaccinia virus MVA-T7-mediated expression of the cDNA in COS7 and Huh-T7 cells, chimeric HAV particles, exposing the foreign epitope gp41 on their surface, were produced. These particles were found to be empty capsids (70S), as judged by immunospecific enzyme linked immunosorbent assay (ELISA) on sucrose gradient fractions and immunoelectron microscopy. The immunological detection of VP1-2A harboring the gp41 epitope of HIV suggests that the 2A domain of HAV is suitable to present foreign antigenic epitopes.