Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1(-/-) mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm(3) in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4(+) or CD8(+) T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifn gamma, Tnf alpha, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1(-/-) mice and that macro- phages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1(-/-) mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1(-/-) mice and may have relevance to relapsing hepatitis A in humans.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.

Weak acid hypochlorous solution (WAHS) is known to have efficacy for inactivating pathogens and to be relatively safe with respect to the live body. Based on these advantages, many animal facilities have recently been introducing WAHS for daily cleaning of animal houses. In this study, we determined the effect of WAHS in inactivating specific pathogens of laboratory rodents and pathogens of opportunistic infection. WAHS with an actual chloride concentration of 60 ppm and a pH value of 6.0 was generated using purpose-built equipment. One volume of mouse hepatitis virus (MHV), Sendai virus, lymphocytic choriomeningitis virus, Bordetella bronchiseptica, Pasteurella pneumotropica, Corynebacterium kutscheri, Staphylococcus aureus, and Pseudomonas aeruginosa was mixed with 9 or 99 volumes of WAHS (×10 and ×100 reaction) for various periods (0.5, 1, and 5 min) at 25°C. After incubation, the remaining infectious viruses and live bacteria were determined by plaque assay or culture. In the ×100 reaction mixture, infectious viruses and live bacteria could not be detected for any of the pathogens examined even with the 0.5-min incubation. However, the effects for MHV, B. bronchiseptica, and P. aeruginosa were variable in the ×10 reaction mixture with the 0.5- and 1-min incubations. Sufficient effects were obtained by elongation of the reaction time to 5 min. In the case of MHV, reducing organic substances in the virus stock resulted in the WAHS being completely effective. WAHS is recommended for daily cleaning in animal facilities but should be used properly in order to obtain a sufficient effect, which includes such things as using a large enough volume to reduce effects of organic substances.

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log(10), respectively, while 70% ethanol reduced the titer by more than 5 log(10). However, WAHS showed more than 5 log(10) reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.

Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Galβ1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.

Previously, we histochemically examined the kidney of the MCC strain of mastomys (Praomys coucha) and found the storage of gangliosides. In the present studies, the lipid-bound sialic acid content of gangliosides in the MCC kidney was about 9- to 14-fold higher than that of the control (MWC strain). In the MCC kidney, sialic acids of male gangliosides were composed of N-acetylneuraminic acid at 91.5%; sialic acids of female gangliosides, however, were composed almost entirely of N-glycolylneuraminic acid. TLC of gangliosides showed that the MCC kidney contained four abundant gangliosides (two gangliosides each in males and females). These gangliosides isolated by HPLC were identified to be GM2(NeuAc) and fucosyl GM1(NeuAc) in the male MCC kidney and GM2(NeuGc) and fucosyl GM1(NeuGc) in the female MCC kidney by secondary ion mass spectrometry, TLC/immunostaining and TLC after enzyme treatments. Although the MCC kidney contained control levels of the activities of beta-N-acetylhexosaminidase, alpha-l-fucosidase, N-acetylgalactosaminyltransferase and fucosyltransferase, the activity of beta-galactosidase in the MCC kidney was increased to 400-500% of that in the MWC kidney. Therefore, we discussed the possibility that in the MCC kidney, GM2 was abundantly produced by the effect of increased beta-galactosidase activity.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.

Persistent LCMV infection in wild-derived MAI/Pas mice housed under conventional conditions remained undetected for a decade, despite periodic health monitoring using dirty-bedding sentinels. When MAI/Pas mice were rederived by embryo transfer, recipient mothers produced antiLCMV antibodies, which first revealed the presence of the virus in the colony. Before this information was obtained, MAI/Pas mice had been shipped to another facility, undergone cesarean rederivation there, and been introduced into the recipient barrier. The foster mothers of rederived pups were LCMV-negative according to enzyme-linked immunosorbent assay, but sera of both cesarean-rederived MAI/Pas mice and their foster mothers were positive for LCMV infection by immunofluorescent assay (IFA). LCMV was isolated from the MAI/Pas mice, and its genomic RNA was sequenced. Examination of animal technicians in contact with LCMV-infected mice and of other mouse samples by IFA or a reverse transcriptase-polymerase chain reaction test (or both) revealed that neither the workers nor other animals had been infected with LCMV. Experimental data showed that LCMV transmission from persistently infected mice to naïve ones occurred only after direct contact of animals housed in the same cage. This experience demonstrates the importance of careful viral monitoring in the transfer of laboratory rodents between institutions, the limitation of dirty-bedding sentinels for detection of LCMV infection, and the inadequacy of cesarean rederivation for elimination of enzootic LCMV infection. 111

Certain low‐molecular‐weight substrate analogs act both as in vitro competitive inhibitors of lysosomal hydrolases and as intracellular enhancers (chemical chaperones) by stabilization of mutant proteins. In this study, we performed oral administration of a chaperone compound N‐octyl‐4‐epi‐β‐valienamine to GM1‐gangliosidosis model mice expressing R201C mutant human β‐galactosidase. A newly developed neurological scoring system was used for clinical assessment. N‐Octyl‐4‐epi‐β‐valienamine was delivered rapidly to the brain, increased β‐galactosidase activity, decreased ganglioside GM1, and prevented neurological deterioration within a few months. No adverse effect was observed during this experiment. N‐Octyl‐4‐epi‐β‐valienamine will be useful for chemical chaperone therapy of human GM1‐gangliosidosis. Ann Neurol 2007

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

The usefulness of RT-PCR for the detection of MHV in tissues and feces of experimentally infected animals has been reported, but it was unclear whether the method was also applicable for the detection of MHV during a natural outbreak. Enterotropic infection is considered to be the most common form of natural infection among various forms of MHV infection. In this paper, RT-nested PCR was performed to detect MHV excreted in the feces during an outbreak in an immunocompromised A/WySnJ mouse colony. The expected bands were amplified after nested PCR from 20 fecal samples out of 37. These results showed that RT-nested PCR could be applicable for the diagnosis for MHV natural infection.

In our laboratory animal facility, Sendai virus (HVJ) contamination occurred in a negative flow rack used for experimental infection with 4 strains of mouse-adapted influenza virus (Inf.V). Anti-HVJ antibody (Ab) was detected in 35/42 mice in the rack. To specify the strain of Inf.V contaminated with HVJ, experimental infection was performed by using A, B and D strains of Inf.V in each vinyl isolator. Anti-HVJ Ab was detected in all mice infected with A strain at day 28 post-infection. As a result of experimental infection with A strain of Inf.V which was treated with anti-HVJ mouse serum, the virus suspension was determined not to contain HVJ and allowed for experimental use in our facility, Since then, HVJ contamination has not occurred in our facility.

The spike (S) protein of a non-fusogenic murine coronavirus, MHV-2, was compared to that of a variant, MHV-2f, with fusion activity. Two amino acids differed between The S proteins of these viruses; one was located in the signal sequence (amino acid 12) and the other in the putative cleavage site (amino acid 757). To determine which one of these amino acid changes is important for the alteration of fusogenicity, chimeric S proteins between MHV-2 and -2f were constructed and expressed in DBT cells by a vaccinia virus expression system. The results revealed that one amino acid change (Ser to Arg) at position 757 is responsible for the acquisition of fusogenicity of the MHV-2f S protein. This change also altered the susceptibility to proteolytic cleavage of the MHV-2 S protein which was originally uncleavable. We concluded that the non-fusogenic activity of MHV-2 results from the lack of cleavage of its S protein.

Mastomys (Praomys coucha) is a small murine rodent native in Africa. The present study was undertaken to investigate the developmental time course of mastomys embryos in vivo and the rate of development in vitro. Mastomys embryos developing in vivo exhibited a long 2-cell stage, which lasted from late Day 1 through Day 3. At Day 4, most embryos developed to the 8-cell or morula stages and at Day 5, all embryos reached the blastocyst stage. The attachment of embryos to the endometrium began at Day 5, as revealed by histological examinations. The development in vitro was investigated in different media with three inbred strains and an F1 hybrid. When 1-cell embryos from inbred mastomys were cultured in Whitten's medium (WM), none of them (n=61) developed beyond the 2-cell stage. Embryos of the same strains collected at Day 2 (2-cells) reached the blastocyst stage (46%, 31/67) in the same medium. Meanwhile, 1-cell embryos from F1 hybrid mastomys developed to the blastocyst stage in WM (13%, 13/71), indicating the presence of a strong heterosis effect. CZB medium, which lacks glucose and contains glutamine and EDTA, supported the development of 1-cell inbred embryos to the blastocyst stage (24%, 17/70). Supplementation of glutamine, but not EDTA, to WM stimulated the development of 1-cells to the blastocyst stage (22%, 11/51). These findings indicate that the development of mastomys embryos in vitro is both strain and medium dependent and that glutamine may play a crucial role in early development of mastomys embryos.

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

The spike (S) protein of a nonfusogenic murine coronavirus, MHV-2, was compared to the S protein of a variant with fusion activity, MHV-2f. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence and the other was in the putative cleavage site. The amino acid at position 12 in the signal sequence was S in MHV-2 and C in MHV-2f. The amino acid sequence of the cleavage site of MHV-2 was HRARS, while that of MHV-2f was HRARR, showing one amino acid replacement at position 757. In DBT cells infected with MHV-2, the S protein was not cleaved, while the S protein of MHV-2f was cleaved. The S protein of MHV-2f expressed in a transient vaccinia virus expression system was cleaved and was fusogenic in contrast to the nonfusogenic activity of uncleaved MHV-2 S protein. Because the signal sequence is assumed to be removed from the mature S protein soon after synthesis, and because the S protein of MHV-2 was expressed on the cell surface in the same way as the S protein of MHV-2f, the difference in the signal sequence seemed to have had little effect on the transportation and the fusion activity of the S protein. These results showed that MHV-2 does not fuse cells due to the lack of cleavage of its S protein. This conclusion differs from studies on the activity of syncytium formation by the S proteins of fusogenic MHV-JHM and -A59 strains. Possible reasons for these differences in fusion activity are discussed.

The renal proximal tubular epithelium of MCC strain of mastomys (Praomys coucha) exhibited a number of cytoplasmic vacuoles after conventional paraffin-embedding procedures. These vacuoles were strongly PAS-positive in cryostat sections. Ultrastructurally, they were double membrane-bound structures filled with myelin figures and acid phosphatase-positive electron-dense matrix. Immunofluorescent microscopy revealed that these structures contained GM(2) ganglioside. Other tissues or organs were histologically normal. Mating experiments indicated that the ganglioside storage in MCC mastomys is inherited as an autosomal recessive trait.