Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein.

UNLABELLED: We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE: AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1.

Herpes simplex virus-1 (HSV-1), the prototype of the α-herpesvirus family, causes life-long infections in humans. Although generally associated with various mucocutaneous diseases, HSV-1 is also involved in lethal encephalitis. HSV-1 entry into host cells requires cellular receptors for both envelope glycoproteins B (gB) and D (gD). However, the gB receptors responsible for its broad host range in vitro and infection of critical targets in vivo remain unknown. Here we show that non-muscle myosin heavy chain IIA (NMHC-IIA), a subunit of non-muscle myosin IIA (NM-IIA), functions as an HSV-1 entry receptor by interacting with gB. A cell line that is relatively resistant to HSV-1 infection became highly susceptible to infection by this virus when NMHC-IIA was overexpressed. Antibody to NMHC-IIA blocked HSV-1 infection in naturally permissive target cells. Furthermore, knockdown of NMHC-IIA in the permissive cells inhibited HSV-1 infection as well as cell-cell fusion when gB, gD, gH and gL were coexpressed. Cell-surface expression of NMHC-IIA was markedly and rapidly induced during the initiation of HSV-1 entry. A specific inhibitor of myosin light chain kinase, which regulates NM-IIA by phosphorylation, reduced the redistribution of NMHC-IIA as well as HSV-1 infection in cell culture and in a murine model for herpes stromal keratitis. NMHC-IIA is ubiquitously expressed in various human tissues and cell types and, therefore, is implicated as a functional gB receptor that mediates broad HSV-1 infectivity both in vitro and in vivo. The identification of NMHC-IIA as an HSV-1 entry receptor and the involvement of NM-IIA regulation in HSV-1 infection provide an insight into HSV-1 entry and identify new targets for antiviral drug development.

Detection of a novel herpesvirus from bats in the Philippines.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

Physical interaction between bovine viral diarrhea virus nonstructural protein 4A and adenosine deaminase acting on RNA (ADAR).

Bovine viral diarrhea virus (BVDV) is a positive-sense RNA virus known to produce double-stranded RNA (dsRNA) during its replication in the cytoplasm. Extended dsRNA duplexes can be hyperedited by adenosine deaminase acting on RNA (ADAR), which catalyzes adenosine (A)-to-inosine (I) editing. A-to-I editing has been reported for various viruses. A number of cellular antiviral defense strategies are stimulated by dsRNA, and this may involve hyperediting of dsRNA by ADARs, followed by targeted cleavage by cytoplasmic endonucleases. Here, we identify ADAR as a binding partner of BVDV NS4A in vitro and in vivo and show that the N-terminal domain of NS4A is the ADAR-binding domain. We also show that ADAR has an inhibitory effect on BVDV replication when overexpressed in BVDV-infected bovine cells. Our findings suggest a role of NS4A in the interaction of BVDV with ADAR that favors virus replication.

Attenuation of an influenza A virus due to alteration of its hemagglutinin-neuraminidase functional balance in mice.

Influenza A viruses possess two surface glycoproteins, hemagglutinin (HA), which binds to sialic-acid-containing receptors, and neuraminidase (NA), which removes sialic acid from host cells. It is well established that the HA-NA functional balance regulates the efficiency of virus replication. Here, we selected a plaque variant of the WSN (H1N1) strain that grew better than the wild-type virus in NA-expressing MDCK cell culture. A reverse genetics study revealed that the single mutation HA E190K, which occurs infrequently in naturally isolated H1N1 viruses, was responsible for the phenotype of this variant. Receptor assays indicated that this mutation did not affect the receptor specificity of HA but enhanced its receptor-binding affinity, resulting in altered HA-NA functional balance relative to that of the wild-type virus. We also found that this variant replicated in nasal turbinates at an equivalent level but in lungs at a lower level compared with wild-type virus, demonstrating its attenuation in mice. Together, our data demonstrated the importance of the HA-NA functional balance for influenza virus replication in an in vivo biological setting.

Genomic and serological detection of bat coronavirus from bats in the Philippines.

Bat coronavirus (BtCoV) is assumed to be a progenitor of severe acute respiratory syndrome (SARS)-related coronaviruses. To explore the distribution of BtCoVs in the Philippines, we collected 179 bats and detected viral RNA from intestinal or fecal samples by RT-PCR. The overall prevalence of BtCoVs among bats was 29.6 %. Phylogenetic analysis of the partial RNA-dependent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others belonged to the genus Betacoronavirus. Western blotting revealed that 66.5 % of bat sera had antibodies to BtCoV. These surveys suggested the endemic presence of BtCoVs in the Philippines.

Detection of bat coronaviruses from Miniopterus fuliginosus in Japan.

Bats have great potential as reservoirs for emerging viruses such as severe acute respiratory syndrome-coronavirus. In this study, bat coronaviruses (BtCoVs) were detected by RT-PCR from intestinal and fecal specimens of Miniopterus fuliginosus breeding colonies in Wakayama Prefecture caves, where we previously identified bat betaherpesvirus 2. Two primer sets were used for the detection of BtCoV: one was for the RNA-dependent RNA polymerase (RdRp) region and the other was for the spike (S) protein region. Eleven and 73% of intestinal and fecal specimens, respectively, were positive for RdRp region, and 2 and 40% of those were positive for S protein region. Sequencing and phylogenetic analysis showed that the detected BtCoV belonged to the group 1 (alpha) coronaviruses. These data suggest that BtCoV is endemic in M. fuliginosus in Japan.

Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines.

BACKGROUND: Ebolaviruses induce lethal viral hemorrhagic fevers (VHFs) in humans and non-human primates, with the exceptions of Reston virus (RESTV), which is not pathogenic for humans. In human VHF cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the IgG responses to nucleoprotein (NP) and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein (GP1,2) are the major indicator of protective immunity. On the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against NP and GP1,2 in RESTV-infected cynomolgus macaques is still unclear. In this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the RESTV epizootic in 1996 in the Philippines to expand our knowledge on the immune responses in naturally RESTV-infected non-human primates. RESULTS: The antibody responses were analyzed using IgG-ELISA, an indirect immunofluorescent antibody assay (IFA), and a pseudotyped VSV-based neutralizing (NT) assay. Antigen-capture (Ag)-ELISA was also performed to detect viral antigens in the serum specimens. We found that the anti-GP1,2 responses, but not the anti-NP responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the RESTV-infected cynomolgus macaques. Additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as IFNγ, IL8, IL-12, and MIP1α, in the convalescent phase sera. CONCLUSIONS: These results imply that both the antibody response to GP1,2 and the proinflammatory innate responses play significant roles in the recovery from RESTV infection in cynomolgus macaques.

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.

A single-amino-acid substitution in herpes simplex virus 1 envelope glycoprotein B at a site required for binding to the paired immunoglobulin-like type 2 receptor alpha (PILRalpha) abrogates PILRalpha-dependent viral entry and reduces pathogenesis.

Paired immunoglobulin-like type 2 receptor α (PILRα) is a herpes simplex virus 1 (HSV-1) entry receptor that associates with O-glycans on HSV-1 envelope glycoprotein B (gB). Two threonine residues (Thr-53 and Thr-480) in gB, which are required for the addition of the principal gB O-glycans, are essential for binding to soluble PILRα. However, the role of the two threonines in PILRα-dependent viral entry remains to be elucidated. Therefore, we constructed a recombinant HSV-1 carrying an alanine replacement of gB Thr-53 alone (gB-T53A) or of both gB Thr-53 and Thr-480 (gB-T53/480A) and demonstrated that these mutations abrogated viral entry in CHO cells expressing PILRα. In contrast, the mutations had no effect on viral entry in CHO cells expressing known host cell receptors for HSV-1 gD, viral entry in HL60 cells expressing myelin-associated glycoprotein (MAG) (another HSV-1 gB receptor), viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. These results support the hypothesis that gB Thr-53 and Thr-480 as well as gB O-glycosylation, probably at these sites, are critical for PILRα-dependent viral entry. Interestingly, following corneal inoculation in mice, the gB-T53A and gB-T53/480A mutations significantly reduced viral replication in the cornea, the development of herpes stroma keratitis, and neuroinvasiveness. The abilities of HSV-1 to enter cells in a PILRα-dependent manner and to acquire specific carbohydrates on gB are therefore linked to an increase in viral replication and virulence in the experimental murine model.

Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step.

Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.

Novel betaherpesvirus in bats.

Because bats are associated with emerging zoonoses, identification and characterization of novel viruses from bats is needed. Using a modified rapid determination system for viral RNA/DNA sequences, we identified a novel bat betaherpesvirus 2 not detected by herpesvirus consensus PCR. This modified system is useful for detecting unknown viruses.

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.

Herpes simplex virus 1 protein kinase Us3 phosphorylates viral envelope glycoprotein B and regulates its expression on the cell surface.

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the -3 and -2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.

Entry of herpes simplex virus 1 and other alphaherpesviruses via the paired immunoglobulin-like type 2 receptor alpha.

Herpes simplex virus 1 (HSV-1) enters cells either via fusion of the virion envelope and host cell plasma membrane or via endocytosis, depending on the cell type. In the study reported here, we investigated a viral entry pathway dependent on the paired immunoglobulin-like type 2 receptor alpha (PILRalpha), a recently identified entry coreceptor for HSV-1 that associates with viral envelope glycoprotein B (gB). Experiments using inhibitors of endocytic pathways and ultrastructural analyses of Chinese hamster ovary (CHO) cells transduced with PILRalpha showed that HSV-1 entry into these cells was via virus-cell fusion at the cell surface. Together with earlier observations that HSV-1 uptake into normal CHO cells and those transduced with a receptor for HSV-1 envelope gD is mediated by endocytosis, these results indicated that expression of PILRalpha produced an alternative HSV-1 entry pathway in CHO cells. We also showed that human and murine PILRalpha were able to mediate entry of pseudorabies virus, a porcine alphaherpesvirus, but not of HSV-2. These results indicated that viral entry via PILRalpha appears to be conserved but that there is a PILRalpha preference among alphaherpesviruses.

Differences in the regulatory and functional effects of the Us3 protein kinase activities of herpes simplex virus 1 and 2.

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.

Molecular cloning and expression analysis of bat toll-like receptors 3, 7 and 9.

In this study, cDNA of Toll-like receptors (TLR) 3, 7 and 9 were synthesized and completely sequenced. The coding regions of cDNA for bat TLR3, TLR7 and TLR9 were 2,718, 3,150 and 3,090 bp in length, respectively. The open reading frames encoded 905, 1,049 and 1,029 amino acids for TLR3, TLR7 and TLR9, respectively. The nucleotide sequences, predicted amino acid sequences and predicted domain structures of the three bat TLRs had high homology with those of other mammals. In addition, the expression profiles of each TLR in main organs were analyzed. Expression of TLR3 was highest in the liver, whereas the expressions of TLR7 and TLR9 were highest in the spleen.

[Current situation and future preventive measures of foot-and-mouth disease in Japan].

Foot-and-mouth disease caused by Foot-and-mouth disease virus (FMDV) is a severe and acute vesicular disease of cloven-hoofed animals including cattle, pigs, sheep and goats. As FMDV is highly contagious and causes productivity losses among infected animals, outbreaks of the disease are a primary animal health concern worldwide. In April, 2010, the disease reoccurred in Miyazaki prefecture in 10 years. Compared to the outbreak in 2000 in which no infection among pigs was observed, a total of 292 infected farms have been involved in this outbreak, and infected animals (37,412 cattle, 42 water buffalos, 174,132 pigs, 14 goats, and 8 sheep) were culled and buried. Vaccination was decided to reduce the speed of virus spreading. Finally a total of 76,756 heads of vaccinated animals were also slaughtered. The outbreak has continued for 2.5 months, and the ban on animal movements have been eased 3 months after the first occurrence. As several factors for disease spreading have been rumored, I would like to note this point and discuss future preventive measures.

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.