A comprehensive approach for evaluating the virucidal performance of domestic laundry detergents under practical conditions.

AIMS: We aimed to develop a method to assess the virucidal performance of domestic laundry in a lab-scale washing machine (Rotawash) based on EN 17658. METHODS AND RESULTS: For method development, virus recovery was investigated after drying on cotton carriers for three test viruses murine norovirus (MNV), modified vaccinia virus Ankara (MVA), and bovine coronavirus (BCoV), followed by washing simulations in flasks and Rotawash. MNV and MVA demonstrated sufficient recovery from carriers after drying and washing (up to 40°C and 60 min). BCoV exhibited lower recovery, indicating less relevance as a test virus. Rotawash efficacy tests conducted with MNV, a resistant, non-enveloped virus, showed limited efficacy of a bleach-free detergent, aligning with results from a domestic washing machine. Rotawash washes achieved higher reductions in infectious virus titers than suspension tests, indicating the role of washing mechanics in virus removal. CONCLUSIONS: This study established a practical method to test the virucidal efficacy of laundry detergents in Rotawash, simulating domestic washing.

A Novel Marsupial Hepatitis A Virus Corroborates Complex Evolutionary Patterns Shaping the Genus Hepatovirus.

The discovery of highly diverse nonprimate hepatoviruses illuminated the evolutionary origins of hepatitis A virus (HAV) ancestors in mammals other than primates. Marsupials are ancient mammals that diverged from other Eutheria during the Jurassic. Viruses from marsupials may thus provide important insight into virus evolution. To investigate Hepatovirus macroevolutionary patterns, we sampled 112 opossums in northeastern Brazil. A novel marsupial HAV (MHAV) in the Brazilian common opossum (Didelphis aurita) was detected by nested reverse transcription-PCR (RT-PCR). MHAV concentration in the liver was high, at 2.5 × 109 RNA copies/g, and at least 300-fold higher than those in other solid organs, suggesting hepatotropism. Hepatovirus seroprevalence in D. aurita was 26.6% as determined using an enzyme-linked immunosorbent assay (ELISA). Endpoint titers in confirmatory immunofluorescence assays were high, and marsupial antibodies colocalized with anti-HAV control sera, suggesting specificity of serological detection and considerable antigenic relatedness between HAV and MHAV. MHAV showed all genomic hallmarks defining hepatoviruses, including late-domain motifs likely involved in quasi-envelope acquisition, a predicted C-terminal pX extension of VP1, strong avoidance of CpG dinucleotides, and a type 3 internal ribosomal entry site. Translated polyprotein gene sequence distances of at least 23.7% from other hepatoviruses suggested that MHAV represents a novel Hepatovirus species. Conserved predicted cleavage sites suggested similarities in polyprotein processing between HAV and MHAV. MHAV was nested within rodent hepatoviruses in phylogenetic reconstructions, suggesting an ancestral hepatovirus host switch from rodents into marsupials. Cophylogenetic reconciliations of host and hepatovirus phylogenies confirmed that host-independent macroevolutionary patterns shaped the phylogenetic relationships of extant hepatoviruses. Although marsupials are synanthropic and consumed as wild game in Brazil, HAV community protective immunity may limit the zoonotic potential of MHAV.IMPORTANCE Hepatitis A virus (HAV) is a ubiquitous cause of acute hepatitis in humans. Recent findings revealed the evolutionary origins of HAV and the genus Hepatovirus defined by HAV in mammals other than primates in general and in small mammals in particular. The factors shaping the genealogy of extant hepatoviruses are unclear. We sampled marsupials, one of the most ancient mammalian lineages, and identified a novel marsupial HAV (MHAV). The novel MHAV shared specific features with HAV, including hepatotropism, antigenicity, genome structure, and a common ancestor in phylogenetic reconstructions. Coevolutionary analyses revealed that host-independent evolutionary patterns contributed most to the current phylogeny of hepatoviruses and that MHAV was the most drastic example of a cross-order host switch of any hepatovirus observed so far. The divergence of marsupials from other mammals offers unique opportunities to investigate HAV species barriers and whether mechanisms of HAV immune control are evolutionarily conserved.

Evolutionary origins of hepatitis A virus in small mammals.

Hepatitis A virus (HAV) is an ancient and ubiquitous human pathogen recovered previously only from primates. The sole species of the genus Hepatovirus, existing in both enveloped and nonenveloped forms, and with a capsid structure intermediate between that of insect viruses and mammalian picornaviruses, HAV is enigmatic in its origins. We conducted a targeted search for hepatoviruses in 15,987 specimens collected from 209 small mammal species globally and discovered highly diversified viruses in bats, rodents, hedgehogs, and shrews, which by pairwise sequence distance comprise 13 novel Hepatovirus species. Near-complete genomes from nine of these species show conservation of unique hepatovirus features, including predicted internal ribosome entry site structure, a truncated VP4 capsid protein lacking N-terminal myristoylation, a carboxyl-terminal pX extension of VP1, VP2 late domains involved in membrane envelopment, and a cis-acting replication element within the 3D(pol) sequence. Antibodies in some bat sera immunoprecipitated and neutralized human HAV, suggesting conservation of critical antigenic determinants. Limited phylogenetic cosegregation among hepatoviruses and their hosts and recombination patterns are indicative of major hepatovirus host shifts in the past. Ancestral state reconstructions suggest a Hepatovirus origin in small insectivorous mammals and a rodent origin of human HAV. Patterns of infection in small mammals mimicked those of human HAV in hepatotropism, fecal shedding, acute nature, and extinction of the virus in a closed host population. The evolutionary conservation of hepatovirus structure and pathogenesis provide novel insight into the origins of HAV and highlight the utility of analyzing animal reservoirs for risk assessment of emerging viruses.

Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release.

Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.

Cellular localization and effects of ectopically expressed hepatitis A virus proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC.

In the course of hepatitis A virus (HAV) infections, the seven nonstructural proteins and their intermediates are barely detectable. Therefore, little is known about their functions and mechanisms of action. Ectopic expression of the presumably membrane-associated proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC allowed the intracellular localization of these proteins and their possible function during the replication cycle of HAV to be investigated. In this study, we used rhesus monkey kidney cells, which are commonly used for cell culture experiments, and human liver cells, which are the natural target cells. We detected specific associations of these proteins with distinct membrane compartments and the cytoskeleton, different morphological alterations of the respective structures, and specific effects on cellular functions. Besides comparable findings in both cell lines used with regard to localization and effects of the proteins examined, we also found distinct differences. The data obtained identify so far undocumented interactions with and effects of the HAV proteins investigated on cellular components, which may reflect unknown aspects of the interaction of HAV with the host cell, for example the modification of the ERGIC (ER-Golgi intermediate compartment) structure, an interaction with lipid droplets and lysosomes, and inhibition of the classical secretory pathway.

Mutational modifications of hepatitis A virus proteins 2B and 2C described for cell culture-adapted and attenuated virus are present in wild-type virus populations.

Studies have identified certain mutations in the 2B and 2C proteins of hepatitis A virus (HAV) as being essential for efficient growth in cultured cells, and it is assumed that these mutations contribute to the attenuated phenotype. We found that mutations supporting cell culture growth already exist in wild-type HAV populations. This suggests that these variants are not entirely generated de novo but are selected from the wild-type population. In a prolonged case of hepatitis A, we found that sequences associated with cell culture adaptation predominated later in infection. This might suggest selection of an attenuated virus population during a prolonged clinical infection.

NF-κB activation induced by hepatitis A virus and Newcastle disease virus occurs by different pathways depending on the structural pattern of viral nucleic acids.

NF-κB is activated by hepatitis B virus and hepatitis C virus and is assumed to contribute to viral persistence, leading to the development of hepatocellular cancer by inhibition of apoptosis mediated by cytotoxic T cells. Whether hepatitis A virus (HAV), which does not cause chronic infection, activates NF-κB is a topic of controversy. Here, we confirm that HAV activates NF-κB and show that HAV enhances the activation of NF-κB by poly(I-C), but it inhibits the activation of NF-κB by Newcastle disease virus (NDV), a paramyxovirus. In addition, HAV inhibits NF-κB activation induced by overexpressed MAVS (mitochondrial antiviral signaling protein). We conclude from these findings that NF-κB induction occurs in cells infected with HAV by dsRNA, independently of mitochondrial-transduced RIG-I/MDA-5 signaling, whereas the induction of NF-κB in cells infected by NDV is mediated by RIG-I signaling, independenly of viral dsRNA. This is supported by experiments in which the different RNA inducers of RIG-I and MDA-5 are sequestered and which also show that poly(I-C) and HAV, but not NDV, are functionally equivalent in inducing NF-κB activity. Furthermore, we demonstrate that HAV interferes with the protein kinase R (PKR) activity and PKR activation induced by dsRNA, and that HAV-induced activation of NF-κB therefore does not take place via the PKR-induced pathway. As assumed for hepatitis B and C virus infections, NF-κB activation could attenuate the effects of cytotoxic T cells and may contribute to prolonged as well as relapsing courses of hepatitis A.

Impact of concentration, temperature and pH on the virucidal activity of alcohols against human adenovirus.

BACKGROUND: Adenoviruses belong to the stable nonenveloped viruses playing an important role in healthcare-associated infections mainly causing respiratory infections and epidemic keratoconjunctivitis. Hand disinfection with alcoholic preparations is therefore one of the most important measures to prevent such viral infections in hospitals and other medical settings. METHODS: The inactivation of adenovirus type 5 by ethanol, 1- and 2-propanol, and 2 commercially available hand disinfectants was examined at different concentrations, temperatures, and pH-values. RESULTS: For ethanol and 1-propanol the maximum virus-inactivating properties after 30 seconds exposure were found at a concentration of 60%-70% and 50%-60%, respectively, whereas with 2-propanol no activity was observed. The virucidal activity of all alcohols and the 2 hand disinfectants examined was increased when raising the temperature from 20°C to 25°C. By increasing the pH value to 9, a strong improvement of the activity of ethanol, 1-propanol and 1 hand disinfectant was observed, whereas pH lowering resulted in decrease of activity. CONCLUSIONS: These results demonstrate the importance of physical parameters in the inactivation of adenoviruses by alcohols and will help to improve measures to reduce adenovirus transmission in healthcare settings.

The role of immunoglobulin A in prolonged and relapsing hepatitis A virus infections.

Hepatitis A virus (HAV) infections result in different courses of the disease, varying between normal, prolonged and relapsing. However, the reason for these heterogeneous clinical appearances is not understood. As HAV-anti-HAV IgA immunocomplexes (HAV-IgA) infect hepatocytes, IgA was postulated as a carrier supporting hepatotropic transport of HAV, and it was speculated that this carrier mechanism contributes to the various clinical outcomes. In this study, the IgA-carrier mechanism was investigated in a mouse model. We show that HAV-IgA immunocomplexes efficiently reached the liver not only in HAV-seronegative mice, but also, and this is in contrast to free-HAV particles, in immunized HAV-seropositive animals. This IgA-mediated transport of HAV to the liver in the presence of immunity depended on the stage of development of the immune response. We conclude that over a period of several weeks after infection, anti-HAV IgA is able to promote an enterohepatic cycling of HAV, resulting in continuous endogenous reinfections of the liver. Our experiments indicate that highly avid IgG antibodies, which are present at later times of the infection, can terminate the reinfections. However, the endogenous reinfections in the presence of a developing neutralizing immunity might contribute to prolonged as well as to relapsing courses of HAV infections. Furthermore, the results show that serum IgA may act as an infection protracting factor.

ABO blood groups and the risk of SARS-CoV-2 infection.

There is no doubt that genetic factors of the host play a role in susceptibility to infectious diseases. An association between ABO blood groups and SARS-CoV-2 infection as well as the severity of COVID-19 has been suggested relatively early during the pandemic and gained enormously high public interest. It was postulated that blood group A predisposes to a higher risk of infection as well as to a much higher risk of severe respiratory disease and that people with blood group O are less frequently and less severely affected by the disease. However, as to the severity of COVID-19, a thorough summary of the existing literature does not support these assumptions in general. Accordingly, at this time, there is no reason to suppose that knowledge of a patient's ABO phenotype should directly influence therapeutical decisions in any way. On the other hand, there are many data available supporting an association between the ABO blood groups and the risk of contracting SARS-CoV-2. To explain this association, several interactions between the virus and the host cell membrane have been proposed which will be discussed here.

Genetic diversity of Dengue virus serotypes circulating among Aedes mosquitoes in selected regions of northeastern Nigeria.

The flaviviruses are mosquito borne pathogens that continue to pose a considerable public health risk to animals and humans. The members of this group includes, Dengue virus (DENV), Yellow fever virus (YVF), Japanese encephalitis virus (JEV), West Nile virus (WEV) and Zika virus (ZKV). The DENV mosquito vector is endemic to tropical and subtropical climates, placing ∼40% of the world's population at direct risk of dengue infection. Currently, in Nigeria the status of DENV serotypes circulating among mosquito vectors is unknown. Our study was designed to identify and characterize the DENV serotypes circulating in Aedes mosquito populations collected in selected sites in Nigeria. The mosquitoes were collected and identified morphologically to species level using colored identification keys of Rueda. Generally, each species identified was tested in pools of 20 individuals of each Aedes species. RT-PCR and semi nested PCR were used to detect DENV serotypes in mosquitoes and characterized using Sanger sequencing methods. The results showed that DENV serotypes were detected in 58.54% (24/41) of the pools of Aedes mosquitoes from Mubi, Numan and Yola screened. All DENV1-4 serotypes were detected in Ae. aegypti. While DENV 1, 2 and 4 were detected in Ae. albopictus. And only DENV 2 was detected in Ae. galloisi with DENV4 serotype being reported for the first time in Nigeria. DENV2 (37.8%) was the most detected serotypes, while double and triple co-infections of serotypes were detected in 24.4% of the pools. Phylogenetic analysis revealed a strong evolutionary relatedness of DENV serotypes in our study with that of South and Southeast Asia, North America, and other African countries. This is the first reports on the natural DENV serotypes co-infection among Aedes species pools in Nigeria, which can create possible interaction with other flaviviruses causing animal and human diseases. In addition, our study postulates the possible linkage between DENV serotypes infection and human febrile flu-like disease burden being experienced by host communities in northeastern Nigeria.

The mitotic spindle: linking teratogenic effects of Zika virus with human genetics?

BACKGROUND: Recently, an association between Zika virus infection and microcephaly/ocular findings was found to be reasonable e.g. because of the demonstration that the virus was found in the brain of a fetus after presumed maternal infection. Although there is no proof yet for a causal relationship, for an appropriate risk calculation efforts are urgently needed to either establish or disprove this assumption. PRESENTATION OF THE HYPOTHESIS: On the basis of inherited syndromes combining microcephaly with ocular findings similar to those associated with Zika infections, we have hypothesized that the impairment of the proper function of the mitotic apparatus is a possible mechanism by which Zika can exert teratogenic effects. TESTING THE HYPOTHESIS: A bundle of well-known cytogenetic and molecular-cytogenetic methods (e.g. formation of micronuclei, chromosomal lagging, immunofluorescence of centrosomes) to evaluate proper function, maintenance, and establishment of the mitotic spindle poles can be applied on infected cells. Also, the viral proteins can be tested for their possible interaction with proteins encoded by genes involved in inherited syndromes with microcephaly and ocular findings resembling those in presumed cases of intrauterine ZIKV infection. IMPLICATIONS OF THE HYPOTHESIS: Once proved, this hypothesis allows for a targeted approach into mechanisms of possible relevance as e.g. if different strains of the virus are implicated in the teratogenic effects to the same or a different extent.

The role of surface functionalization of colloidal alumina particles on their controlled interactions with viruses.

Materials that interact in a controlled manner with viruses attract increasing interest in biotechnology, medicine, and environmental technology. Here, we show that virus-material interactions can be guided by intrinsic material surface chemistries, introduced by tailored surface functionalizations. For this purpose, colloidal alumina particles are surface functionalized with amino, carboxyl, phosphate, chloropropyl, and sulfonate groups in different surface concentrations and characterized in terms of elemental composition, electrokinetic, hydrophobic properties, and morphology. The interaction of the functionalized particles with hepatitis A virus and phages MS2 and PhiX174 is assessed by virus titer reduction after incubation with particles, activity of viruses conjugated to particles, and imaged by electron microscopy. Type and surface density of particle functional groups control the virus titer reduction between 0 and 99.999% (5 log values). For instance, high sulfonate surface concentrations (4.7 groups/nm(2)) inhibit attractive virus-material interactions and lead to complete virus recovery. Low sulfonate surface concentrations (1.2 groups/nm(2)), native alumina, and chloropropyl-functionalized particles induce strong virus-particle adsorption. The virus conformation and capsid amino acid composition further influence the virus-material interaction. Fundamental interrelations between material properties, virus properties, and the complex virus-material interaction are discussed and a versatile pool of surface functionalization strategies controlling virus-material interactions is presented.

Innate and adaptive immune responses against picornaviruses and their counteractions: An overview.

Picornaviruses, small positive-stranded RNA viruses, cause a wide range of diseases which is based on their differential tissue and cell type tropisms. This diversity is reflected by the immune responses, both innate and adaptive, induced after infection, and the subsequent interactions of the viruses with the immune system. The defense mechanisms of the host and the countermeasures of the virus significantly contribute to the pathogenesis of the infections. Important human pathogens are poliovirus, coxsackievirus, human rhinovirus and hepatitis A virus. These viruses are the best-studied members of the family, and in this review we want to present the major aspects of the reciprocal effects between the immune system and these viruses.

Comparison of mechanical properties of normal and malignant thyroid cells.

Cancer is a disease of uncontrolled cell proliferation causing approximately 13% of deaths worldwide. Cancer cell mechanics is currently an important topic of investigation in cancer diagnostics as a possible tool to distinguish malignant cells from normal cells in addition to increasing our understanding of pathophysiology of the disease. Our study, based on Atomic Force Microscopy (AFM) measurements on cells, shows that malignant thyroid cells are 3- to 5-fold softer in comparison to primary normal thyroid cells depending on duration between cell seeding and AFM experiments. These results reveal cultivation period as an important factor that influences cell mechanics and which must be considered when comparing cells. Investigation of actin cytoskeleton by fluorescent labelling revealed differences in organization of actin between malignant and normal thyroid cells, which may be directly contributing to alteration of cell mechanics in cancer cells.

Hepatitis A virus protein 2B suppresses beta interferon (IFN) gene transcription by interfering with IFN regulatory factor 3 activation.

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of retinoic acid-inducible gene I-mediated and melanoma differentiation-associated gene 5-mediated beta interferon (IFN-beta) gene expression. This study showed that this is due to an interaction of HAV with mitochondrial antiviral signalling protein (MAVS)-dependent signalling, in which the viral non-structural protein 2B and the protein intermediate 3ABC recently suggested in this context seem to be involved, cooperatively affecting the activities of MAVS and the kinases TANK-binding kinase 1 (TBK1) and the inhibitor of NF-kappaB kinase epsilon (IKKepsilon). In consequence, interferon regulatory factor 3 (IRF-3) is not activated. As IRF-3 is necessary for IFN-beta transcription, inhibition of this factor results in efficient suppression of IFN-beta synthesis. This ability might be of vital importance for HAV, which is an exceptionally slow growing virus sensitive to IFN-beta, as it allows the virus to establish infection and maintain virus replication for a longer period of time.

IgA-coated particles of Hepatitis A virus are translocalized antivectorially from the apical to the basolateral site of polarized epithelial cells via the polymeric immunoglobulin receptor.

Although Hepatitis A virus (HAV) is transmitted by the faecal-oral route, its target for replication is the liver. Little is known of its interactions with cells of the gastrointestinal tract, and it is not known by which mechanisms HAV crosses the intestinal epithelium. In this study, it is shown that HAV associated with IgA is translocated from the apical to the basolateral compartment of polarized epithelial cells via the polymeric immunoglobulin receptor by IgA-mediated reverse transcytosis. The relevance of this mechanism, by which HAV-IgA complexes may overcome the intestinal barrier and contribute to infections of the liver, results from the fact that HAV-IgA complexes are infectious for hepatocytes and that significant amounts of intestinal HAV-IgA are present during acute infections, which are also partly transmitted. Besides supporting the primary infection, this mechanism may play a role in relapsing infections by establishing an enterohepatic cycle for HAV.

Hepatitis A virus suppresses RIG-I-mediated IRF-3 activation to block induction of beta interferon.

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of double-stranded RNA (dsRNA)-induced beta interferon (IFN-beta) gene expression. In this report, we show that this is due to an interaction of HAV with the intracellular dsRNA-induced retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway upstream of the kinases responsible for interferon regulatory factor 3 (IRF-3) phosphorylation (TBK1 and IKKepsilon). In consequence, IRF-3 is not activated for nuclear translocation and gene induction. In addition, we found that HAV reduces TRIF (TIR domain-containing adaptor inducing IFN-beta)-mediated IRF-3 activation, which is part of the Toll-like receptor 3 signaling pathway. As IRF-3 is necessary for IFN-beta transcription, inhibition of this factor results in efficient suppression of IFN-beta synthesis. This ability of HAV seems to be of considerable importance for HAV replication, as HAV is not resistant to IFN-beta, and it may allow the virus to establish infection and preserve the sites of virus production in later stages of the infection.

Occurrence of hepatitis A virus genotype III in Germany requires the adaptation of commercially available diagnostic test systems.

BACKGROUND: A blood donation, obtained in 2003 in Germany during the preseroconversion diagnostic window period of a hepatitis A virus (HAV) infection, tested HAV-negative by commercially available HAV reverse transcription-polymerase chain reaction (RT-PCR) detection assays. STUDY DESIGN AND METHODS: The virus responsible for this infection was identified as HAV genotype IIIA by characterization of the nearly complete genome sequence. RESULTS: Thereby, this HAV variant, which was named strain HMH, was detected in Germany for the first time. Because the commercially available HAV RNA detection systems failed to detect this genotype, a real-time RT-PCR kit was developed that allows quantification and detection of all HAV genotypes. The first nearly full-length nucleotide sequence so far available for HAV genotype IIIA is also provided. CONCLUSION: This case demonstrates that owing to the genetic variability of HAV, constant monitoring and adaptation of the diagnostic nucleic acid assays are required to guarantee the safety of blood and blood products.

Adaptation of primate cell-adapted hepatitis A virus strain HM175 to growth in guinea pig cells is independent of mutations in the 5' nontranslated region.

Previous studies of hepatitis A virus (HAV) genotypes after adaptation of wild-type virus to growth in cell cultures of primate origin identified determinants for growth in cell culture in the viral 2B and 2C protein-coding regions of the genome and demonstrated that an increased growth efficiency in a particular cell line was achieved by subsequent mutations in the 5' nontranslated region (5'NTR). The results reported in this study demonstrate that the passage of HAV adapted to primate BS-C-1 cells in guinea pig cells resulted in increased growth efficiency in the rodent cells and decreased growth efficiency in BS-C-1 cells. This adaptation occurred without mutation in the 5'NTR, but the viral 2B and 2C proteins seem to play a role during adaptation to the new environment, as one mutation occurred in each protein. Although the data presented here do not clearly identify which region of the viral genome underwent mutations to improve the interaction of the viruses with guinea pig proteins, they do confirm that the 5'NTR is not the only region responsible for providing host cell-specific information.