Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

Intracerebral Inoculation of Mouse-Passaged Saffold Virus Type 3 Affects Cerebellar Development in Neonatal Mice.

Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice by using SAFV passaged in the cerebella of neonatal BALB/c mice. The virus titer in the cerebellum increased following the inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via the formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. Compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and greater replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or the passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants. IMPORTANCE Saffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained an in vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life.

The contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of S proteins and incorporation of S protein into virus-like particles.

The cytoplasmic tails of some coronavirus (CoV) spike (S) proteins contain an endoplasmic reticulum retrieval signal (ERRS) that can retrieve S proteins from the Golgi to the endoplasmic reticulum (ER); this process is thought to accumulate S proteins at the CoV budding site, the ER-Golgi intermediate compartment (ERGIC), and to facilitate S protein incorporation into virions. However, we showed previously that porcine epidemic diarrhoea CoV S proteins lacking the ERRS were efficiently incorporated into virions, similar to the original virus. Thus, the precise role of the ERRS in virus assembly remains unclear. Here, the roles of the S protein ERRS in severe acute respiratory syndrome CoV (SARS-CoV) intracellular trafficking and S incorporation into virus-like particles (VLPs) are described. Intracellular trafficking and indirect immunofluorescence analysis suggested that when M protein was present, wild-type S protein (wtS) could be retained in the pre- and post-medial Golgi compartments intracellularly and co-localized with M protein in the Golgi. In contrast, mutant S protein lacking the ERRS was distributed throughout the ER and only partially co-localized with M protein. Moreover, the intracellular accumulation of mutant S protein, particularly at the post-medial Golgi compartment, was significantly reduced compared with wtS. A VLP assay suggested that wtS that reached the post-medial compartment could be returned to the ERGIC for subsequent incorporation into VLPs, while mutant S protein could not. These results suggest that the ERRS of SARS-CoV contributes to intracellular S protein accumulation specifically in the post-medial Golgi compartment and to S protein incorporation into VLPs.

Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.

Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus.

Possible involvement of infection with human coronavirus 229E, but not NL63, in Kawasaki disease.

Although human coronavirus (HCoV)-NL63 was once considered a possible causative agent of Kawasaki disease based on RT-PCR analyses, subsequent studies could not confirm the result. In this study, this possibility was explored using serological tests. To evaluate the role of HCoV infection in patients with Kawasaki disease, immunofluorescence assays and virus neutralizing tests were performed. Paired serum samples were obtained from patients with Kawasaki disease who had not been treated with γ-globulin. HCoV-NL63 and two antigenically different isolates of HCoV-229E (ATCC-VR740 and a new isolate, Sendai-H) were examined as controls. Immunofluorescence assays detected no difference in HCoV-NL63 antibody positivity between the patients with Kawasaki disease and controls, whereas the rate of HCoV-229E antibody positivity was higher in the patients with Kawasaki disease than that in controls. The neutralizing tests revealed no difference in seropositivity between the acute and recovery phases of patients with Kawasaki disease for the two HCoV-229Es. However, the Kawasaki disease specimens obtained from patients in recovery phase displayed significantly higher positivity for Sendai-H, but not for ATCC-VR740, as compared to the controls. The serological test supported no involvement of HCoV-NL63 but suggested the possible involvement of HCoV-229E in the development of Kawasaki disease.

Neuropathogenesis of a mouse-adapted porcine epidemic diarrhea virus infection in suckling mice.

A mouse-adapted porcine epidemic diarrhea virus, MK-p10, showed higher neurovirulence in suckling mice than a non-adapted MK strain. There was no difference in virus growth, whereas clear differences between these two virus infections existed in the type of target cells infected, the spread of virus and the cytokine levels produced in the brain. In the early phase of infection, neurons, astrocytes and neural progenitor cells were infected by MK-p10, whereas neural progenitor cells were the only target cells infected by MK. On days 4-5 post-inoculation, MK-p10 antigens were distributed in a number of neurons in a wide area of the brain; however, antigens were restricted in MK infection. In moribund mice in both infection groups, viral antigens were found in a wide area of the brain. The wide spectrum of initial target cells following MK-p10 infection, as well as its faster spread in the brain, may be evidence of enhanced virulence in suckling mice.

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry.

The type II transmembrane protease TMPRSS2 activates the spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) on the cell surface following receptor binding during viral entry into cells. In the absence of TMPRSS2, SARS-CoV achieves cell entry via an endosomal pathway in which cathepsin L may play an important role, i.e., the activation of spike protein fusogenicity. This study shows that a commercial serine protease inhibitor (camostat) partially blocked infection by SARS-CoV and human coronavirus NL63 (HCoV-NL63) in HeLa cells expressing the receptor angiotensin-converting enzyme 2 (ACE2) and TMPRSS2. Simultaneous treatment of the cells with camostat and EST [(23,25)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester], a cathepsin inhibitor, efficiently prevented both cell entry and the multistep growth of SARS-CoV in human Calu-3 airway epithelial cells. This efficient inhibition could be attributed to the dual blockade of entry from the cell surface and through the endosomal pathway. These observations suggest camostat as a candidate antiviral drug to prevent or depress TMPRSS2-dependent infection by SARS-CoV.

Hemagglutination mediated by the spike protein of cell-adapted bovine torovirus.

Bovine torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity.

Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M-S co-localization.

The endodomain of several coronavirus (CoV) spike (S) proteins contains palmitylated cysteine residues and enables co-localization and interaction with the CoV membrane (M) protein. Depalmitylation of mouse hepatitis virus S proteins abolished this interaction, resulting in the failure of S incorporation into virions. In contrast, an immunofluorescence assay (IFA) showed that depalmitylated severe acute respiratory syndrome coronavirus (SCoV) S proteins still co-localized with the M protein in the budding site. Here, we determined the ability of depalmitylated SCoV S mutants to incorporate S into virus-like particles (VLPs). IFA confirmed that all SCoV S mutants co-localized with the M protein intracellularly. However, the mutants lacking two cysteine residues (C(1234/1235)) failed to incorporate S into VLPs. This indicated that these palmitylated cysteines are essential for S incorporation, but are not involved in S co-localization mediated by the M protein. Our findings suggest that M-S co-localization and S incorporation occur independently of one another in SCoV virion assembly.

Differences in neutralizing antigenicity between laboratory and clinical isolates of HCoV-229E isolated in Japan in 2004-2008 depend on the S1 region sequence of the spike protein.

Human coronavirus (HCoV) is a causative agent of the common cold. Although HCoV is highly prevalent in the world, studies of the genomic and antigenic details of circulating HCoV strains have been limited. In this study, we compared four Japanese isolates with the standard HCoV-229E strain obtained from ATCC (ATCC-VR740) by focusing on the spike (S) protein, a major determinant of neutralizing antigen and pathogenicity. The isolates were found to have nucleotide deletions and a number of sequence differences in the S1 region of the S protein. We compared two of the Japanese isolates with the ATCC-VR740 strain by using virus-neutralizing assays consisting of infectious HCoV-229E particles and vesicular stomatitis virus (VSV)-pseudotyped virus carrying the HCoV-229E S protein. The two clinical isolates (Sendai-H/1121/04 and Niigata/01/08) did not react with antiserum to the ATCC-VR740 strain via the neutralizing test. We then constructed a pseudotype VSV-harboured chimeric S protein with the ATCC S1 and Sendai S2 regions or that with Sendai S1 and ATCC S2 regions and compared them by a neutralization test. The results revealed that the difference in the neutralizing antigenicity depends on the S1 region. This different antigenic phenotype was also confirmed by a neutralizing test with clinically isolated human sera. These results suggest that the HCoV-229E viruses prevalent in Japan are quite different from the laboratory strain ATCC-VR740 in terms of the S sequence and neutralization antigenicity, which is attributed to the difference in the S1 region.

Role of proteases in the release of porcine epidemic diarrhea virus from infected cells.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhea, requires a protease(s) for multicycle replication in cultured cells. However, the potential role of proteases in the infection process remains unclear. In order to explore this, we used two different approaches: we infected either Vero cells in the presence of trypsin or Vero cells that constitutively express the membrane-associated protease TMPRSS2 (Vero/TMPRSS2 cells). We found that PEDV infection was enhanced, and viruses were efficiently released into the culture fluid, from Vero cells infected in the presence of protease, while in cells without protease, the virus grew, but its release into the culture fluid was strongly hampered. Cell-to-cell fusion of PEDV-infected cells and cleavage of the spike (S) protein were observed in cells with protease. When infected Vero cells were cultured for 3 days in the absence of trypsin but were then treated transiently with trypsin, infectious viruses were immediately released from infected cells. In addition, treatment of infected Vero/TMPRSS2 cells with the protease inhibitor leupeptin strongly blocked the release of virus into the culture fluid. Under electron microscopy, PEDV-infected Vero cells, as well as PEDV-infected Vero/TMPRSS2 cells treated with leupeptin, retained huge clusters of virions on their surfaces, while such clusters were rarely seen in the presence of trypsin and the absence of leupeptin in Vero and Vero/TMPRSS2 cells, respectively. Thus, the present study indicates that proteases play an important role in the release of PEDV virions clustered on cells after replication occurs. This unique observation in coronavirus infection suggests that the actions of proteases are reminiscent of that of the influenza virus neuraminidase protein.

Reduction of Cell Fusion by Deletion in the Hypervariable Region of the Spike Protein of Mouse Hepatitis Virus.

Deletions in the spike gene of mouse hepatitis virus (MHV) produce several variants with diverse biological characteristics, highlighting the significance of the spike gene in viral pathogenesis. In this study, we characterized the JHM-X strain, which has a deletion in the hypervariable region (HVR) of the spike gene, compared with the cl-2 strain, which has a full spike gene. Cytopathic effects (CPEs) induced by the two strains revealed that the size of the CPE produced by cl-2 is much greater than that produced by JHM-X in delayed brain tumor (DBT) cells. Thus, this finding explains the greater fusion activity of cl-2 than JHM-X in cultured cells, and we speculate that the deletion region of the spike protein is involved in the fusion activity differences. In contrast with the fusion activity, a comparison of the virus growth kinetics revealed that the titer of JHM-X was approximately 100 times higher than that of cl-2. We found that the deletion region of the spike protein was involved in fusion activity differences, whereas cl-2 produced significantly higher luciferase activity than JHM-X upon similar expression levels of the spike protein. However, the reason behind the growth difference is still unknown. Overall, we discovered that deletion in the HVR of the spike gene could be involved in the fusion activity differences between the two strains.

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.

Role of mouse hepatitis virus (MHV) receptor murine CEACAM1 in the resistance of mice to MHV infection: studies of mice with chimeric mCEACAM1a and mCEACAM1b.

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD(50)) of 10(2.5) PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10(5) PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10(5) PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.

Spongiform degeneration induced by neuropathogenic murine coronavirus infection.

Soluble receptor-resistant mutant 7 (ssr7) is isolated from a highly neurovirulent mouse hepatitis virus (MHV) JHMV cl-2 strain (cl-2). srr7 exhibits lower virulence than its maternal strain in infected mice, which is typically manifested in a longer lifespan. In this study, during the course of infection with srr7, small spongiotic lesions became apparent at 2 days post-inoculation (pi), they spread out to form spongiform encephalopathy by 8 to 10 days pi. We recently reported that the initial expressions of viral antigens in the brain are detected in the infiltrating monocyte lineage and in ependymal cells. Here, we demonstrate that the next viral spread was observed in glial fibrillary acidic protein-positive cells or nestin-positive progenitor cells which take up positions in the subventricular zone (SVZ). From this restricted site of infection in the SVZ, a large area of gliosis extended deep into the brain parenchyma where no viral antigens were detected but vacuolar degeneration started at 48 h pi of the virus. The extremely short incubation period compared with other experimental models of infectious spongiform degeneration in the brain would provide a superior experimental model to investigate the mechanism of spongiotic lesions formation.

[Coronaviruses].

Coronaviruses contain positive-stranded RNA with ca. 30 kb as a genome, which is wrapped by the envelope, and constitute Nidovirales together with Arteriviridae. The feature of viruses in Nidovirales is the unique structure of the mRNA set, called 3' co-terminal nested set. Coronaviruses have several to more than 10 different species of subgenomic mRNA and generally only the OFR located in the 5' end of each mRNA is translated. The 5' 20 kb of the coronavirus genome or mRNA-1 consists of two ORFs, 1a and 1b, between that there is a unique RNA structure called pseudoknot. From mRNA-1, 1a as well as 1a+1b are translated; the latter 1a+1b results from the translation due to ribosomal frame-shifting facilitated by the pseudoknot structure. From those two proteins, totally 16 proteins are produced as a result of auto-cleavage by the proteases included in la protein. Those proteins exhibit different functions, such as RNA-dependent RNA polymerase, helicase, proteases and proteins that regulate cellular functions, mRNAs smaller than mRNA-2 translate in general the structural proteins, nucleocapsid (N) protein, spike (S) protein, integrated membrane (M) protein and envelope (E) proteins. Those proteins assemble to the vesicles located from ER to Golgi (ER Golgi intermediate compartment) and virions bud into the vesicles. Those virions are released from infected cells via exocytosis.

Recent Progress in Torovirus Molecular Biology.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis.

The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.