Identification and characterization of the genome of a papillomavirus from skin lesions of four-toed hedgehogs (Atelerix albiventris).

The present study describes the clinical and pathological characteristics of skin lesions in two four-toed hedgehogs (Atelerix albiventris). We performed inverse PCR to identify the genome of papillomavirus (PV) in the skin lesions and subsequently sequenced the full genome of the virus, which was tentatively named Atelerix albiventris papillomavirus 1 (AalbPV1). The overall sequences of the viral genomes of both four-toed hedgehogs were identical. This study first identified the presence of a novel PV in Japanese four-toed hedgehogs and provided genetic information about this virus.

Identification and characterization of a novel bat polyomavirus in Japan.

A novel polyomavirus (PyV) was identified in the intestinal contents of Japanese eastern bent-wing bats (Miniopterus fuliginosus) via metagenomic analysis. We subsequently sequenced the full genome of the virus, which has been tentatively named Miniopterus fuliginosus polyomavirus (MfPyV). The nucleotide sequence identity of the genome with those of other bat PyVs was less than 80%. Phylogenetic analysis revealed that MfPyV belonged to the same cluster as PyVs detected in Miniopterus schreibersii. This study has identified the presence of a novel PyV in Japanese bats and provided genetic information about the virus.

Novel cyclovirus detected in the intestinal contents of Taiwan squirrels (Callosciurus erythraeus thaiwanensis).

A novel cyclovirus was identified in the intestinal contents of Taiwan squirrels (Callosciurus erythraeus thaiwanensis) collected in Kanagawa prefecture, Japan, by metagenomic analysis, and was named Taiwan squirrel cyclovirus-1 (TsCyV-1). Phylogenetic analysis showed that TsCyV-1 formed a branch separate from other representative cyclovirus strains. TsCyV-1 is considered to be a new species in the genus Cyclovirus because the criteria for demarcation of cyclovirus species is proposed as nucleotide identities <80 %.

The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers.

This study examined the virucidal effects of five types of alcohol-based sanitizers including malic acid and sodium malate, or monoethanolamin, in 58 vol % ethanol (pH 4.0, pH 7.1, pH 11.8), 65 vol % ethanol (pH 4.2), and 75 vol % ethanol (pH 4.4) against murine norovirus (MNV) and feline calicivirus (FCV). The virus titer of MNV was reduced in an ethanol dose-dependent manner under the same pH (about 4.0) condition. Virucidal effect against MNV was correlated with pH when the concentration of ethanol was constant (58 vol %). All the ethanol-based sanitizers provided sufficient virucidal effects against FCV. In conclusion, the virucidal effect of the ethanol-based sanitizer at low concentration of ethanol against norovirus (NoV) is increased when the pH is adjusted to a neutral state.

Identification of amino acid substitutions escaping from a broadly neutralizing monoclonal antibody of feline calicivirus.

Feline calicivirus (FCV) causes upper respiratory tract diseases in cats and has highly variable antigenicity for neutralization of each strain. Neutralizing epitopes of FCV are currently found in the hypervariable region (HVR) in the P2 domain of the major capsid protein VP1. Due to its unique ability to neutralize various FCV strains, 1D7 is a monoclonal antibody that may recognize a novel neutralizing epitope. While other neutralizing epitopes were characterized by producing neutralization-resistant variants, only 1D7-resistant variants could not be obtained, and its epitope has not been identified in the previous studies. In this study, we successfully generated these variants by multiple passaging of the FCV F4 strain in the presence of 1D7 and discovered that several amino acid substitutions (K638N, R662G, and T666I in the P1 domain of VP1) are involved in the decreased binding of 1D7. These substitution sites are also highly conserved among FCV strains compared with the substitution sites of other neutralization-resistant variants found in the HVR. Our results indicate that amino acid substitutions in the P1 domain, which are not responsible for direct interaction with the FCV receptor, are associated with neutralization escape. Since FCV can be conveniently cultured in vitro and the receptor required for infection is known, a detailed analysis of the 1D7 epitope could shed more light on the neutralization mechanism of the epitopes of viruses belonging to the Caliciviridae.

Plasmodium falciparum BAEBL binds to heparan sulfate proteoglycans on the human erythrocyte surface.

Erythrocyte invasion is critical to the pathogenesis and survival of the malarial parasite, Plasmodium falciparum. This process is partly mediated by proteins that belong to the Duffy binding-like family, which are expressed on the merozoite surface. One of these proteins, BAEBL (also known as EBA-140), is thought to bind to glycophorin C in a sialic acid-dependent manner. In this report, by the binding assay between recombinant BAEBL protein and enzyme-treated erythrocytes, we show that the binding of BAEBL to erythrocytes is mediated primarily by sialic acid and partially through heparan sulfate (HS). Because BAEBL binds to several kinds of HS proteoglycans or purified HS, the BAEBL-HS binding was found to be independent of the HS proteoglycan peptide backbone and the presence of sialic acid moieties. Furthermore, both the sialic acid- and HS-dependent binding were disrupted by the addition of soluble heparin. This inhibition may be the result of binding between BAEBL and heparin. Invasion assays demonstrated that HS-dependent binding was related to the efficiency of merozoite invasion. These results suggest that HS functions as a factor that promotes the binding of BAEBL and merozoite invasion. Moreover, these findings may explain the invasion inhibition mechanisms observed following the addition of heparin and other sulfated glycoconjugates.

Caliciviruses induce mRNA of tumor necrosis factor α via their protease activity.

Calicivirus infection in patients and animals is associated with the production of multiple inflammatory cytokines, including tumor necrosis factor α (TNF-α). Here we studied the feline calicivirus (FCV) non-structural proteins and found that the FCV protease was a key factor for TNF-α gene expression in cultured cells. The expression of the TNF-α gene in cells expressing FCV, human norovirus, and rabbit hemorrhagic disease virus protease was compared, revealing that the induction of TNF-α could be a common phenomenon during the infection by the viruses in the Caliciviridae. The level of TNF-α mRNA in the cells expressing mutant proteases that lacked the active site was measured. These data indicate that the protease activity is crucial for TNF-α expression. These findings provide new insight into the induction of inflammation during calicivirus infection.

Bat coronaviruses and experimental infection of bats, the Philippines.

Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription-PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9-1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that non-structural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK- and IKKbeta-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-kappaB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597-623. In addition, overexpression of NS5A inhibited NF-kappaB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-kappaB signalling.

Nosocomial outbreak of serious canine infectious tracheobronchitis (kennel cough) caused by canine herpesvirus infection.

Canine herpesvirus (CHV; Canid herpesvirus 1) is principally a perinatal pathogen of pregnant bitches and newborn pups and secondarily a respiratory tract pathogen of older pups and dogs. Infectious disease of the canine respiratory tract frequently occurs among dogs in groups, in which it is called " infectious tracheobronchitis" (ITB). Mortality from ITB is generally negligible, and the clinical importance of CHV as an ITB pathogen is considered to be low. The present report describes a novel ITB outbreak accompanied by death among aged dogs in an animal medical center. Most inpatient dogs had received medications that could induce immunosuppression. CHV was the only pathogen identified, and several CHV isolates were recovered in cell culture. No other viral pathogens or significant bacterial pathogens were found. Molecular and serological analyses revealed that the causative CHV isolates were from a single source but that none was a peculiar strain when the strains were compared with previous CHV strains. The virus had presumably spread among the dogs predisposed to infection in the center. The present results serve as a warning to canine clinics that, under the specific set of circumstances described, such serious CHV outbreaks may be expected wherever canine ITB occurs.

Suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses.

nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs.

Microarray analysis reveals distinct signaling pathways transcriptionally activated by infection with bovine viral diarrhea virus in different cell types.

Infection with bovine viral diarrhea virus (BVDV) causes different effects depending on its biotype in vitro; cytopathogenic (cp) strains induce apoptosis, type I interferon (IFN), and various stress-mediated responses, whereas non-cytopathogenic (ncp) strains do not. However, comprehensive transcriptional profiles of the cells infected with BVDV are still unknown. Here we performed microarray analysis of BVDV-infected MDBK epithelial cells and bovine fetal muscle (BFM) fibroblast cells. Infection of both cell types with cp BVDV, but not ncp BVDV, stimulated marked up-regulation of numerous genes belonging to diverse functional classes. However, the pattern of gene expression detected in both cell types was highly distinct from each other. Notably, upon cp BVDV infection, BFM cells exhibited marked induction of IFN-stimulated genes (ISGs), whereas MDBK cells characteristically up-regulated endoplasmic reticulum stress-inducible genes, such as tribbles homolog 3 (TRB3), CHOP and asparagine synthase, and showed much weaker induction of ISGs than BFM cells. This study highlights unexpected diversity in the response of different cell types to BVDV infection.

Application of retrovirus-mediated expression cloning for receptor screening of a parasite.

Retrovirus-mediated expression cloning has been applied in both virology and cell biology. Although there is some difficulty in applying this technique to screening for a receptor recognized by an intracellular parasite, we modified the conventional method to identify a putative receptor for the Plasmodium falciparum BAEBL protein. We show that this method is effective in screening for a parasite receptor.

Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome.

Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.

Characterization and application of monoclonal antibodies to bovine viral diarrhea virus nonstructural protein 5A.

Bovine viral diarrhea virus (BVDV) nonstructural protein 5A (NS5A) is one the least studied of the BVDV proteins. Therefore, to develop a tool for unraveling the functions performed by BVDV NS5A, monoclonal antibodies (MAbs) were generated by fusion of myeloma cells with spleen cells from mice immunized with recombinant E. coli-expressed GST-NS5A protein. Two MAbs (1H12 and 2F9) were established on the basis of immunofluorescence and Western blot analysis. Both the MAbs were of IgG1 subclass and recognized an epitope clustered within the N-terminal region of NS5A. Furthermore, the MAb 1H12 was used successfully to detect NS5A protein in BVDV field isolates belonging to genotypes 1 and 2. Temporal expression pattern studies during an infectious cycle revealed that BVDV NS5A could be detected 12-60 h postinfection. Confocal microscopy studies showed a cytoplasmic staining pattern and revealed that NS5A is localized on the endoplasmic reticulum membrane in BVDV infected cells.

Activation of extracellular signal-regulated kinase in MDBK cells infected with bovine viral diarrhea virus.

Our efforts to identify the cellular signaling cascades triggered by bovine viral diarrhea virus (BVDV) infection in MDBK cells revealed marked activation of extracellular signal-regulated kinase 1/2 (ERK). Enhanced phosphorylation of ERK was detected following infection with cytopathogenic (cp) BVDV, but not with noncytopathogenic BVDV. It appears that cp BVDV-induced ERK phosphorylation is caused by oxidative stress, because ERK phosphorylation was inducible by treatment with hydrogen peroxide or serum deprivation and was attenuated by addition of antioxidants. These results indicate that BVDV infection influences the ERK signaling pathway via oxidative stress, depending on the biotype.

Detection of a new bat gammaherpesvirus in the Philippines.

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3' part of the gB gene and the 5' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Inactivation of human norovirus and its surrogate by the disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals.

Human norovirus is one of the major causes of foodborne gastroenteritis, and it can be easily transmitted from infected person, virus-contaminated foods and environmental surfaces. Effective disinfection method is needed to stop the transmission of human norovirus. CAC-717 is a new disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals. We aimed to evaluate the efficacy of CAC-717 against human norovirus. This study used human norovirus derived from fecal specimens and cultured murine norovirus, which is one of the surrogate viruses for human norovirus. The disinfection effect against murine norovirus was estimated by infectivity assay and transmission electron microscopy. The inactivation effect against human norovirus was assessed by reverse transcription polymerase chain reaction. Disinfection effect of CAC-717 against the infectivity of murine norovirus was shown within 100 s after the CAC-717 treatment, presenting the destruction of viral capsids. The treatment of CAC-717 significantly reduced human norovirus genomic RNA (3.25-log reduction) by the presence of the mesoscopic structure of calcium hydrogen carbonate. CAC-717 stably inactivated human norovirus in stool suspensions. The inactivation effect of CAC-717 against human norovirus was less susceptible to organic substances than sodium hypochlorite. CAC-717 would be a useful alternative for disinfecting human norovirus in contaminated environmental surfaces.

Characterization of a novel species of adenovirus from Japanese microbat and role of CXADR as its entry factor.

Recently, bat adenoviruses (BtAdVs) of genus Mastadenovirus have been isolated from various bat species, some of them displaying a wide host range in cell culture. In this study, we isolated two BtAdVs from Japanese wild microbats. While one isolate was classified as Bat mastadenovirus A, the other was phylogenetically independent of other BtAdVs. It was rather related to, but serologically different from, canine adenoviruses. We propose that the latter, isolated from Asian parti-colored bat, should be assigned to a novel species of Bat mastadenovirus. Both isolates replicated in various mammalian cell lines, implying their wide cell tropism. To gain insight into cell tropism of these BtAdVs, we investigated the coxsackievirus and adenovirus receptor (CXADR) for virus entry to the cells. We prepared CXADR-knockout canine kidney cells and found that replication of BtAdVs was significantly hampered in these cells. For confirmation, their replication in canine CXADR-addback cells was rescued to the levels with the original cells. We also found that viral replication was corrected in human or bat CXADR-transduced cells to similar levels as in canine CXADR-addback cells. These results suggest that BtAdVs were able to use several mammalian-derived CXADRs as entry factors.

Characterization of Plasmodium falciparum protein kinase 2.

A sustained elevation of free Ca(2+) is observed on the rupture and release of merozoites of Plasmodium falciparum from the erythrocytes. The immunoelectron micrographs demonstrate that calmodulin is localized in merozoites. To elucidate the Ca(2+) signal of P. falciparum invasion, we attempted to characterize P. falciparum protein kinase 2 (PfPK2), which is homologous to human calcium calmodulin-dependent protein kinase (CaMK). PfPK2 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP; this labeling was then eliminated by phosphatase. This phosphorylation was eliminated when the putative catalytic lysine residue of PfPK2 was replaced with alanine. PfPK2 phosphorylated histone II(AS) as a representative substrate in a Ca(2+)- and calmodulin-dependent manner. Calmodulin antagonists inhibited the phosphorylation of PfPK2 in vitro and markedly decreased the parasitemia of ring forms in an invasion assay, whereas CaMKII-specific inhibitors had no effect. PfPK2 was localized in the merozoites in the culture of P. falciparum. Thus, purified PfPK2 possesses protein kinase activity in a Ca(2+)- and calmodulin-dependent manner and the catalytic lysine of this protein was determined. These data suggest that PfPK2 is the Plasmodium protein kinase expressed in the merozoites during the invasion stage.