Human sapovirus propagation in human cell lines supplemented with bile acids.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log10-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Discovery and Synthesis of Heterocyclic Carboxamide Derivatives as Potent Anti-norovirus Agents.

There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure-activity relationship (SAR) of a series of heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC50)=37 µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC50=24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC50=5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC50 value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.

Myeloid-associated differentiation marker is an essential host factor for human parechovirus PeV-A3 entry.

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

Amplification of all 11 RNA segments of group A rotaviruses based on reverse transcription polymerase chain reaction.

Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double-stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full-length sequences of long RNA segments of rotaviruses by one-time only RT-PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high-fidelity and rapid DNA polymerase in a one-step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS-1, Hochi, 69M, WI61, M37 and SA11-S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.

[Possibilities and the assignments for infection control of norovirus: the verification and the limit of inactivation efficacy by surrogate viruses].

Norovirus (NoV) was isolated from the feces of patients were infected during the outbreak of enteritis in Ohio in the early 1970s. Subsequent experimental studies on volunteers proved that NoV was the enteric virus that caused diarrhea, vomiting, and fever. However, the culture of NoV has been unsuccessful, because the mechanism of NoV propagating in cells, NoV infection routes, and methods for preventing NoV infection are unclear. Therefore, from the 1980s, feline calicivirus (FCV), which is cultivable, has been used mainly in research on virus inactivation as a surrogate virus for NoV. In 2003, a new cultivable calicivirus, the murine norovirus (MNV) was isolated from specific mice colonies, and the virus was classified in the NoV geno-group. MNV has also been used in in vitro studies as a surrogate for NoV. However, MNV does not possess structural variety like NoV and FCV. Therefore, when investigating the effect of chemical disinfectants on surrogates for NoV, it is desirable to experiment with other MNV variants or FCV. In future, if large-scale in vitro culture of NoV is successful, we will be able to compare the effect of virucides on structural denaturalization in NoV and surrogate viruses.

A Human Intestinal Cell Line Suitable for the Propagation of Human Parechovirus Type 1 to 6 with a Clear Cytopathic Effect.

Human parechoviruses (HPeVs) are being increasingly recognized as pathogens that cause mild-to-life-threatening diseases in children and adults. Recently, nucleic acid detection has become the mainstream method for pathogen detection. However, virus isolation is important for virus detection and further virologic characterization studies, and securing human pathogenic virus bioresources. We recently explored conventional cell lines suitable for human sapovirus isolation and accidentally identified a human duodenal cell line, HuTu80, that supported efficient growth of human parechovirus type 3 (HPeV-3) with clear cytopathic effects (CPE). Subsequently, we confirmed that all representative prototype HPeV type 1-6 strains were propagated efficiently in HuTu80 cells with clear CPE within 4 days. Another human ileocecal cell line, HCT-8 (HRT-18), also supports HPeV propagation except for HPeV-3. Titer values in HuTu80 and HCT-8 reached approximately 6.83-8.83 and 6.50-8.17 log10 50% tissue culture infectious dose/50 µL, respectively, when inoculated with multiplicity of infection of 0.0025. Previously reported cell lines likely support HPeV types 1-6 with different efficiency, especially for HPeV-3. In summary, HuTu80 can be used as an additional cell line for HPeV isolation, propagation with a clear CPE to produce a high titer value and for the virus neutralization assays.

Characterization of a Human Sapovirus Genotype GII.3 Strain Generated by a Reverse Genetics System: VP2 Is a Minor Structural Protein of the Virion.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5'-cap structure is essential for the virus' generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Potential risk of SARS-CoV-2 infection among people handling linens used by COVID-19 patients before and after washing.

The risk of SARS-CoV-2 infection when people handle linens is uncertain. We examined the presence of SARS-CoV-2 on linens, in the air, and on personal protective equipment (PPE) to assess potential infection risk among individuals who handle linens used by SARS-CoV-2-infected people. Patients in a hospital and an accommodation facility who tested positive for SARS-CoV-2 participated in this study in 2020. Linen samples before washing or disinfection, rinse water after washing or disinfection, air in the workplace at the hospital and an accommodation facility, and the PPE worn by linen-handling people were tested for SARS-CoV-2 RNA and viable viruses. Among 700 samples from 13 SARS-CoV-2-infected participants and their surrounding environment, SARS-CoV-2 RNA was detected from 14% (52/362) of the linens used by COVID-19 patients (cycle threshold [Ct] value: 33-40). SARS-CoV-2 RNA was detected from 8% (2/26) of rinse water after washing or disinfection, from 15% (16/104) of air samples in the workspace, and from 10% (5/52) of gowns worn by linen-handling people, all with high Ct values (> 36). No SARS-CoV-2 was isolated from any samples. The potential risk of SARS-CoV-2 infection from handling linens used by SARS-CoV-2-infected people exists but appears to below.

[Efficacy of inactivating viruses by photocatalytically reacting nonwoven titanium dioxide fabric].

Titanium dioxide (TiO2) photocatalysis causes oxidative destruction dependent on electrons excited by < or = 400 nm ultraviolet (UV) rays. Many studies have covered the destruction of organics and bacteria and bacteriophage inactivation by photocatalysis. We studied the inactivation by new nonwoven siliconized titanium dioxide fabric of the feline calicivirus F9 (FCV-F9), human adenovirus GB (HAdv3-GB), and influenza A and B virus (A/New Caledonia, B/Shandong, and 5 clinical strains). We spotted 10 microL of viral suspensions containing infectious 5 log10 50% tissue culture doses (TCID50) onto 1 cm2 pieces of TiO2-coated nonwoven control fabric treated or not treated with UV light (lambda(max), 365 nm, 1,100-1,300 microW/cm2). We then measured the virus titers of 50 microL of viral suspension recovered from these fabrics. FCV-F9 and HAdv3-GB infectivity titers were reduced by over 3.5 log10 TCID50 after 30 min of irradiation, but influenza viral titer was reduced to where it was undetectable even without UV irradiation. Comparing individual viral titer reduction due to nonwoven fabric contact without UV irradiation exposure, showed that FCV-F9 and HAdv3-GB titer infectivity was not reduced. In contrast, influenza A and B titer infectivity was reduced to 2 log10 TCID50 after 5 min of contact with the nonwoven fabric and to 3 log10 TCID50 after 30 min of contact. Titers of 6 of 7 influenza A and B strains were reduced by over 4 log10 TCID50 within 30 min. Siliconized TiO2-coated nonwoven fabric thus efficiently inactivated FCV-F9 and HAdV-GB and absorbed influenza viruses.

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan.

Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.

Pathotypes and Drug Susceptibility of Escherichia coli Isolated from Companion Dogs in Japan.

Considering the possibility that Escherichia coli carried by companion dogs could infect owners and human society, we investigated their pathogenicity and drug resistance. E. coli was isolated from stool samples of companion dogs (n = 90) to examine the O-serogroup, virulence genes, and drug susceptibility. The age of dogs ranged from 4 months to 16 years, and they were mainly treated with cefalexin, enrofloxacin, or amoxicillin. A total of 69 samples were positive for E. coli (76% of examined dogs), and the most common O-serogroup was O18 (n = 13). Nine diarrheagenic E. coli, including enteropathogenic E. coli (n = 3), enteroaggregative E. coli (n = 1), and astA-carrying E. coli (n = 5), were isolated. In addition, we isolated 28 E. coli strains resistant to at least one of six antimicrobials, including cephalothin (CET), ceftazidime (CAZ), cefotaxime (CTX), chloramphenicol (CP), fosfomycin (FOM), and norfloxacin (NLFX). The resistance pattern was as follows: CET, n = 16; NLFX, n = 3; CET/CP (resistance to both CET and CP), n = 1; CET/NLFX, n = 1; CET/CAZ/CTX, n = 3; CET/CTX/NLFX, n = 2; CET/CP/NLFX, n = 1; and CET/CAZ/CTX/NLFX, n = 1. Moreover, ten E. coli isolates were found to produce extended-spectrum ß-lactamase (ESBL), including AmpC (n = 4; OUT, O18, O74, and O166), CTX-M-1 (n = 1; O25), CTX-M-9 (n = 4; OUT, O18, O18, and O125), and AmpC/CTX-M-9 (n = 1; OUT) groups. The AmpC-producing E. coli strains included enteropathogenic and astA-carrying E. coli. Our results showed that the human-infectious diarrheagenic E. coli was isolated from some dogs, and some strains exhibited ESBL. Therefore, future studies are needed to investigate the possibility of transmission of these E. coli strains to humans.

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV.

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

A PCR-based protocol for generating West Nile virus replicons.

A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon.

Identification of antibody against porcine coronavirus in human milk.

A total of 239 human milk samples collected from mothers in Chiba City, Japan during 1994-1997 were tested for the presence of anti-coronavirus antibody. Interestingly, twelve human milk samples were positive for IgA against porcine transmissible gastroenteritis coronavirus, and this represented 5%. These findings provided evidence that the interspecies transmission of coronavirus between human and porcine might occur in nature. This report is noteworthy because it is the first, to the best of our knowledge, demonstrating the presence of antibody against porcine transmissible gastroenteritis coronavirus in human milk.

Viral Population Changes during Murine Norovirus Propagation in RAW 264.7 Cells.

Laboratory adaptation of viruses is an essential technique for basic virology research, including the generation of attenuated vaccine strains, although the principles of cell adaptation remain largely unknown. Deep sequencing of murine norovirus (MuNoV) S7 during serial passages in RAW264.7 cells showed that the frequencies of viral variants were altered more dynamically than previously reported. Serial passages of the virus following two different multiplicity of infections gave rise to distinct haplotypes, implying that multiple cell-adaptable sequences were present in the founder population. Nucleotide variants lost during passage were assembled into a viral genome representative of that prior to cell adaptation, which was unable to generate viral particles upon infection in cultured cells. In addition, presence of the reconstructed genome interfered with production of infectious particles from viruses that were fully adapted to in vitro culture. Although the key nucleotide changes dictating cell adaptation of MuNoV S7 viral infection are yet to be elucidated, our results revealed the elaborate interplay among selected sequences of viral variants better adapted to propagation in cell culture. Such knowledge will be instrumental in understanding the processes necessary for the laboratory adaptation of viruses, especially to those without relevant cell culture systems.

A Proposal for a Structural Model of the Feline Calicivirus Protease Bound to the Substrate Peptide under Physiological Conditions.

Feline calicivirus (FCV) protease functions to cleave viral precursor proteins during productive infection. Previous studies have mapped a protease-coding region and six cleavage sites in viral precursor proteins. However, how the FCV protease interacts with its substrates remains unknown. To gain insights into the interactions, we constructed a molecular model of the FCV protease bound with the octapeptide containing a cleavage site of the capsid precursor protein by homology modeling and docking simulation. The complex model was used to screen for the substrate mimic from a chemical library by pharmacophore-based in silico screening. With this structure-based approach, we identified a compound that has physicochemical features and arrangement of the P3 and P4 sites of the substrate in the protease, is predicted to bind to FCV proteases in a mode similar to that of the authentic substrate, and has the ability to inhibit viral protease activity in vitro and in the cells, and to suppress viral replication in FCV-infected cells. The complex model was further subjected to molecular dynamics simulation to refine the enzyme-substrate interactions in solution. The simulation along with a variation study predicted that the authentic substrate and anti-FCV compound share a highly conserved binding site. These results suggest the validity of our in silico model for elucidating protease-substrate interactions during FCV replication and for developing antivirals.

Antiviral effect of theaflavins against caliciviruses.

Caliciviruses are contagious pathogens of humans and various animals. They are the most common cause of viral gastroenteritis in humans, and can cause lethal diseases in domestic animals such as cats, rabbits and immunocompromised mice. In this study, we conducted cytopathic effect-based screening of 2080 selected compounds from our in-house library to find antiviral compounds against three culturable caliciviruses: feline calicivirus, murine norovirus (MNV) and porcine sapovirus (PoSaV). We identified active six compounds, of which two compounds, both related to theaflavins, showed broad antiviral activities against all three caliciviruses; three compounds (abamectin, a mixture of avermectin B1a and B1b; avermectin B1a; and (-)-epigallocatechin gallate hydrate) were effective against PoSaV only; and a heterocyclic carboxamide derivative (BFTC) specifically inhibited MNV infectivity in cell cultures. Further studies of the antiviral mechanism and structure-activity relationship of theaflavins suggested the following: (1) theaflavins worked before the viral entry step; (2) the effect of theaflavins was time- and concentration-dependent; and (3) the hydroxyl groups of the benzocycloheptenone ring were probably important for the anti-calicivirus activity of theaflavins. Theaflavins could be used for the calicivirus research, and as potential disinfectants and antiviral reagents to prevent and control calicivirus infections in animals and humans.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

Development of a novel single step reverse genetics system for feline calicivirus.

The reverse genetics system is a useful tool to generate infectious virus. Feline calicivirus (FCV), a member of the genus Vesivirus in the family Caliciviridae, has a positive sense, single-stranded RNA genome. Two reverse genetics systems have been established for FCV; however, these methods need multi-steps to produce progeny infectious virus. In this study, a novel plasmid-based single step reverse genetics system for FCV has been developed. The plasmid carries FCV F4 strain genomic sequence with an introduced silent mutation. In addition, at the 5'- and 3'-end, a human elongation factor-1α promoter and a cis-acting hepatitis delta virus ribozyme following poly-A, were added, respectively. When the plasmid was transfected into Crandell-Rees feline kidney cells, progeny FCV was generated. The reverse genetics system-derived FCV (rFCV) showed similar growth kinetics and antigenic characteristics and had identical genomic terminals to those of the original FCV F4 strain. The presence of the introduced silent mutation in the rFCV genomic cDNA supported that the progeny virus was originated from the plasmid. This novel FCV reverse genetics system is simple and can be used to evaluate the functions of the viral genome, proteins, and phenotypic characterization of FCV strains in the future.