Torque-mixing magnetic resonance spectroscopy.

A universal, torque-mixing method for magnetic resonance spectroscopy is presented. In analogy to resonance detection by magnetic induction, the transverse component of a precessing dipole moment can be measured in sensitive broadband spectroscopy, here using a resonant mechanical torque sensor. Unlike induction, the torque amplitude allows equilibrium magnetic properties to be monitored simultaneously with the spin dynamics. Comprehensive electron spin resonance spectra of a single-crystal, mesoscopic yttrium iron garnet disk at room temperature reveal assisted switching between magnetization states and mode-dependent spin resonance interactions with nanoscale surface imperfections. The rich detail allows analysis of even complex three-dimensional spin textures. The flexibility of microelectromechanical and optomechanical devices combined with broad generality and capabilities of torque-mixing magnetic resonance spectroscopy offers great opportunities for development of integrated devices.

WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A.

BACKGROUND: We recently described an inherited coagulopathy arising in an inbred colony of WAG/RijYcb rats. The bleeding phenotype, demonstrated by both male and female rats, included periarticular hemorrhage, spontaneous bruising, prolonged bleeding from minor wounds and maternal peripartum deaths. Coagulation testing of affected rats revealed normal prothrombin time but prolongation of activated partial thromboplastin time to twice that of controls. OBJECTIVE: To determine the specific coagulation factor and the underlying genetic defect responsible for the inherited coagulopathy in the WAG/RijYcb rats. RESULTS: Evaluation of individual clotting factor activities revealed that the affected animals had a specific deficiency of factor (F) VIII (FVIII). The FVIII gene (F8) has an autosomal location on chromosome 18 in rats, in contrast to its location on the X chromosome in mice and humans. Sequencing of F8 cDNA led to the identification of a point mutation resulting in a substitution, Leu176Pro, in the A1 domain, that is predicted to disrupt the tertiary structure of the FVIII molecule. Administration of human plasma or human recombinant FVIII corrects the coagulation abnormality in the affected animals. CONCLUSIONS: We have now identified the genetic basis of the hemostatic defect in the WAG/RijYcb rat colony. The larger size of rats relative to mice and the presence of this coagulation defect in both sexes provide a unique model, well-suited to the development of novel therapies for acquired and hereditary FVIII deficiencies.

Persistent Seoul virus infection in Lewis rats.

Mechanistic studies of hantavirus persistence in rodent reservoirs have been limited by the lack of a versatile animal model. This report describes findings from experimental infection of inbred Lewis rats with Seoul virus strain 80-39. Rats inoculated with virus intraperitoneally at 6 days of age became persistently infected without clinical signs. Tissues from Seoul virus-inoculated 6-day-old rats were assessed at 6, 9, and 12 weeks post-inoculation for viral RNA by RT-PCR and in situ hybridization (ISH) and for infectious virus by inoculation of Vero E6 cells. Virus was isolated from lung and kidney of infected rats at 6 weeks and viral RNA was detected in lung, kidney, pancreas, salivary gland, brain, spleen, liver and skin at 6, 9 and 12 weeks. Rats inoculated with Seoul virus intraperitoneally at 10 or 21 days of age became infected without clinical signs but had low to undetectable levels of viral RNA in tissues at 6 weeks post-inoculation. ISH identified vascular smooth muscle and endothelial cells as common sites of persistent infection. Cultured rat smooth muscle cells and to a lesser extent cultured endothelial cells also were susceptible to Seoul virus infection. Pancreatic infection resulted in insulitis with associated hyperglycemia. These studies demonstrate that infant Lewis rats are uniformly susceptible to asymptomatic persistent Seoul virus infection. Additionally, they offer opportunities for correlative in vivo and in vitro study of Seoul virus interactions in host cell types that support persistent infection.

Detection of infectious agents in laboratory rodents: traditional and molecular techniques.

Methods to detect infectious agents in laboratory animals have traditionally been serological and culture based. Molecular methods to detect infectious agents in laboratory animals are being used more routinely. Confusion as to when and how to use molecular methods abounds. In this review, we present a guide to the weaknesses and strengths of using traditional and molecular methods for the detection of infectious agents in laboratory animals.

Dual infection with Pneumocystis carinii and Pasteurella pneumotropica in B cell-deficient mice: diagnosis and therapy.

BACKGROUND AND PURPOSE: The clinical presentation, diagnosis, histopathologic findings, and elimination of dual respiratory tract infection with Pasteurella pneumotropica and Pneumocystis carinii were studied in 100 adult barrier-reared C.B17 and MRL- lpr mice homozygous for a targeted mutation of the JH region of the immunoglobulin heavy chain. METHODS: Necropsy, aerobic bacteriologic culture of hematogenous and pulmonary tissues, histochemical staining of pulmonary tissues, polymerase chain reaction analysis of pulmonary tissues and feces, and viral serologic testing were performed on 19 clinically affected mice and 8 clinically normal mice, then later on antibiotic-treated and caesarian re-derived mice. Therapeutic strategies included sequential administration of trimethoprim/ sulfamethoxazole and enrofloxacin or enrofloxacin administration and caesarian rederivation. RESULTS: Clinically affected mice had diffuse, nonsuppurative, interstitial pneumonia with superimposed pyogranulomatous lobar pneumonia that was detected microscopically. Affected lung tissue yielded pure culture of P. pneumotropica. Aged-matched, clinically normal mice of both genotypes had interstitial histiocytic pneumonia without lobar pneumonia, and P. pneumotropica was not isolated. Histochemical staining of lung tissues from normal and clinically affected mice revealed scattered cysts consistent with P. carinii, principally in the interstitium. Treatment with sulfamethoxazole/trimethoprim and enrofloxacin eliminated bacteriologic detection of P. pneumotropica, decreased mortality from 50% to 6%, and improved breeding performance. CONCLUSION: A successful antibiotic therapy and rederivation approach, incorporating enrofloxacin, cesarian section, and isolator rearing, was developed for B cell-deficient mice with opportunistic infections.

Reverse transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new rat coronavirus strain.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCV) are highly infectious and spread rapidly through laboratory rat colonies, causing sneezing, nasal and ocular discharges, photophobia, and cervical swelling. Current diagnostic methods include serologic testing and histologic examination. During a recent rat coronavirus outbreak, we tested a rapid, noninvasive method of RCV diagnosis that involved use of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to detect RCV RNA on cages housing infected rats. METHODS: The RT-PCR was used to detect RCV RNA in tissues from infected rats and on cages housing infected rats and to amplify portions of the RCV N, M, and S genes for molecular characterization. RESULTS: The RT-PCR detected RCV RNA on cages and in tissues from infected rats. The RCV-NJ N gene is most closely related to the MHV-Y N gene. The M proteins of RCV-NJ and RCV-SDA are 99% homologous, and the six RCV S protein fragments are 97 to 100% homologous. CONCLUSIONS: Use of RT-PCR with cage-swab specimens was capable of diagnosing RCV infection in and viral excretion from rats. Additionally, molecular characterization of the N, M, and S genes of RCV-NJ provided baseline information that can be used in performing further epidemiologic studies.

Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCVs) are common natural pathogens of rats that cause clinical illness, necrosis, and inflammation of respiratory, salivary, and lacrimal organs. The aim of the study was to determine whether antigenically different strains of RCV vary in their pathogenic potential in rats. METHODS: Neutralization groups were identified by use of RCV strain-specific antisera. Sprague Dawley rats were inoculated oronasally with RCV-SDA, RCV-BCMM, or RCV-W. Histologic examination, immunohistochemical analysis, and reverse transcriptase-polymerase chain reaction analysis were performed on tissues from infected rats. RESULTS: Clinical illness was not evident in any of the inoculated rats. The RCV-SDA strain caused mild lesions in the exorbital gland of one rat. The RCV-BCMM strain caused severe lesions in the Harderian and parotid glands and mild lesions in the exorbital glands, lungs, and nasal mucosa. The RCV-W strain caused severe lesions in the Harderian, exorbital, and parotid glands and mild lesions in the submandibular glands, lungs, and nasal mucosa. The RNA concentration was highest in the Harderian, parotid, and exorbital glands of RCV-BCMM- and RCV-W-infected rats at postinoculation day 7. CONCLUSIONS: Although RCV-SDA, RCV-BCMM, and RCV-W caused different degrees and patterns of lesions, neutralization groups are not useful for predicting the pathogenic potential of a new RCV isolate.

Interactions of enterotropic mouse hepatitis viruses with Bgp2 receptor proteins.

Enterotropic mouse hepatitis virus (MHV) infections are limited to the intestinal mucosa, rarely disseminate to other tissues and cause disease only in neonatal mice. The role of virus-host cell receptor interactions in the limited tissue tropism of enterotropic MHV infections is unclear. Previous studies have shown that enterotropic MHV-Y can infect BHK cells stably transfected with either the MHVR or the mmCGM2 receptor gene. In contrast, enterotropic MHV-RI infects BHK cells stably transfected with the MHVR but not the mmCGM2 receptor gene. Studies to determine whether MHV-Y and -RI can utilize the Bgp2 receptor isoform were performed. Both MHV-Y and -RI infected Vero cells transiently transfected with the Bgp2 receptor gene, though only MHV-Y infected CHO cells stably transfected with the Bgp2 receptor gene. Additionally, pretreatment with anti-MHVR monoclonal antibody (CC1) did not prevent MHV-Y and -RI infection of CMT93 cells. In contrast, pretreatment with CC1 prevented MHV-A59 infection of CMT93 cells. It is likely that MHV-Y and -RI use the Bgp2 receptor to infect CC1 pretreated CMT93 cells, since CMT93 cells are known to possess high levels of the Bgp2 receptor mRNAs, but it is also possible that they use an unidentified receptor.

Growth characteristics and protein profiles of prototype and wild-type rat coronavirus isolates grown in a cloned subline of mouse fibroblasts (L2p.176 cells).

Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolates. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.

Optimization of in vitro growth conditions for enterotropic murine coronavirus strains.

Enterotropic mouse hepatitis virus (MHV) strains have been difficult to grow in cell culture. In an attempt to develop an efficient in vitro cultivation system for enterotropic MHV strains (MHV-RI and MHV-Y), 8 murine cell lines were inoculated with MHV-RI- or MHV-Y-infected infant mouse intestinal homogenates and screened for the production of cytopathic effects. MHV-RI and MHV-Y consistently produced cytopathic effects only in J774A.1 cells. Both strains produced titers of > 10(6) TCID50/ml in subsequent passages in J774.1 cells. MHV strains-1, -3, -A59, -JHM, -S and -DVIM also produced high-titer viral stocks in J774A.1 cells. Therefore J774A.1 cells are the first cells found that support the replication of these 8 enterotropic and respiratory MHV strains. After passage in J774A.1 cells, MHV-RI and MHV-Y could infect previously non-susceptible cell lines (17Cl-1, CMT-93, N18 and NCTC 1469), though cytopathic effects were often negligible with MHV-RI. MHV-Y, but not MHV-RI, grew in L2(Percy) cells. Using L2(Percy) cells, an agarose overlay and Giemsa staining, MHV-Y could be quantified by plaque assay. Infant mouse bioassay, plaque assays and cell culture infections were compared for their sensitivity in detecting MHV-Y in infected intestinal homogenates and cell supernatants.

Molecular characterization of the S proteins of two enterotropic murine coronavirus strains.

Enterotropic strains of murine coronaviruses (MHV-Y and MHV-RI) differ extensively in their pathogenesis from the prototypic respiratory strains of murine coronaviruses. In an effort to determine which viral proteins might be determinants of enterotropism, immunoblots of MHV-Y and MHV-RI virions using anti-S, -N and -M protein-specific antisera were performed. The uncleaved MHV-Y and MHV-RI S proteins migrated slightly faster than the MHV-A59 S protein. The MHV-Y S protein was inefficiently cleaved. The MHV-Y, MHV-RI and MHV-A59 N and M proteins showed only minor differences in their migration. The S genes of MHV-Y and MHV-RI were cloned, sequenced and found to encode 1361 and 1376 amino acid long proteins, respectively. The presence of several amino acids changes upstream from the predicted cleavage site of the MHV-Y S protein may contribute its inefficient cleavage. A high degree of homology was found between the MHV-RI and MHV-4 S proteins, whereas the homology between the MHV-Y S protein and the S proteins of other MHV strains was much lower. These results indicate that the enterotropism of MHV-RI and MHV-Y may be determined by different amino acid changes in the S protein and/or by changes in other viral proteins.

Characterization of the S protein of enterotropic murine coronavirus strain-Y.

The pathogenesis of enterotropic murine coronavirus strain MHV-Y differs extensively from that of prototypic respiratory strains of murine coronaviruses. The S protein of MHV-Y was characterized as a first step towards identifying viral determinants of enterotropism. Immunoblots of MHV-Y virions using anti-S protein specific antiserum revealed that the MHV-Y S protein was inefficiently cleaved. The MHV-Y S gene was cloned and sequenced. It encodes a protein predicted to be 1361 amino acids long. The presence of several amino acids changes within and surrounding the predicted cleavage site of the MHV-Y S protein may contribute to its inefficient cleavage.

Multiple receptor-dependent steps determine the species specificity of HCV-229E infection.

Human coronavirus (HCV)-229E causes disease only in humans and grows in human cells and in cells of other species that express recombinant human aminopeptidase N (hAPN), the receptor for HCV-229E. We compared the species specificity of HCV-229E infection with the species specificity of virus binding using immunofluorescence, assay of virus yields, fluorescence activated cell sorting and a monoclonal antibody directed against hAPN that blocks infection. We found that HCV-229E binds to intestinal brush border membranes (BBM) and to membranes of cell lines from cats, dogs, pigs, and humans, however the virus only infects two of these species. HCV-229E will not bind to BBM or to membranes from cell lines derived from hamster or mice. Animal coronaviruses related to HCV-229E, including FIPV, CCV, and TGEV bind to cell membranes from cats, dogs, cows, pigs and humans (but not mice), while each virus infects cells from only a subset of these species. Infectious genomic HCV-229E RNA, can infect cells of all of these species. These data suggest that the species-specificity of infection for this serogroup of coronaviruses is determined at the levels of virus binding and penetration. Since binding of viral spike glycoprotein to cellular receptors is not the only limiting factor, we suggest that one or more steps associated with virus penetration may determine the species specificity of infection with the HCV-229E serogroup of coronaviruses.

Enterotropic strains of mouse coronavirus differ in their use of murine carcinoembryonic antigen-related glycoprotein receptors.

Enterotropic mouse hepatitis virus strains (MHV-RI and MHV-Y) replicate in the intestine and rarely disseminate to other tissues, unlike respiratory MHV strains. Murine carcinoembryonic antigen-related glycoproteins (MHVR and mmCGM2) are expressed in many murine tissues and serve as receptors for respiratory MHV strains. To assess the role of receptors in the limited tissue tropism of enterotropic MHV strains, the permissiveness of MHVR- and mmCGM2-expressing cell lines and peritoneal exudate cells from BALB/c and SJL mice for MHV-RI and MHV-Y replication was determined. MHVR-transfected BHK cells were susceptible to infection with both MHV-RI and MHV-Y. Additionally, the anti-MHVR monoclonal antibody CC1 blocked MHV-RI and MHV-Y infection. mmCGM2-transfected BHK cells were susceptible to infection with MHV-Y, but not MHV-RI. Peritoneal exudate cells from BALB/c mice were susceptible to infection with MHV-Y and MHV-RI, whereas peritoneal exudate cells from SJL mice were susceptible to infection with MHV-Y but not MHV-RI. These results indicate that MHV-RI probably uses a different receptor than other MHV strains to infect SJL mice and that receptors are probably not the primary determinant of the limited tissue tropism of enterotropic MHV strains.

Development and optimization of plaque assays for rat coronaviruses.

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaque-reduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.

The cellular and molecular pathogenesis of coronaviruses.

Coronaviruses cause a wide spectrum of diseases in humans and animals but generally fall into two classes, with respiratory or enteric tropisms. Mouse hepatitis virus (MHV) and rat coronaviruses are the viruses most frequently encountered in the laboratory animal setting. This review focuses primarily on the cellular and molecular aspects of MHV pathogenesis. The high mutation and recombination rates of coronaviruses lead to a diverse, ever-changing population of MHV strains. The spike (S) protein is the most variable coronavirus protein and is responsible for binding to cell surface receptors, inducing cell fusion and humoral and cellular immunity. Differences within the S protein of different MHV strains have been linked to their variable tropisms. Since immunity to MHV is strain-specific, seropositive mice can be reinfected with different strains of MHV. Natural infections with MHV are acute, with persistence occurring at the population level, not within an individual mouse, unless it is immunocompromised. Age, genotype, immunologic status of the mouse, and MHV strain influence the type and severity of disease caused by MHV. Interference with research by MHV has been reported primarily in the fields of immunology and tumor biology and may be a reflection of MHV's capacity to grow in several types of immune cells. While many methods are available to diagnose coronavirus infection, serologic tests, primarily ELISA and IFA, are the most commonly used. MHV is best managed on a preventive basis. Elimination of MHV from a population requires cessation of breeding and halting the introduction of naive mice into the population.

Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.

Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions.

A temperature-sensitive mutant of poliovirus, VP2-103, was isolated and characterized. A single nucleotide change, resulting in the substitution of glutamine for arginine at amino acid 76 of the capsid protein VP2, prevented the maturation of virions at the nonpermissive temperature. Particles indistinguishable from the previously elusive provirions were observed; these particles have been proposed to be penultimate in virion morphogenesis. Cleavage of VP0 into VP2 and VP4, the products found in mature virions, was not observed in VP2-103-infected cells at the nonpermissive temperature. The cleavage of VP0 in wild-type poliovirus-infected cells is dependent on RNA packaging; this reaction has been postulated to be autocatalytic. The existence of RNA-containing provirionlike particles in VP2-103-infected cells shows that RNA packaging can be uncoupled from VP0 cleavage.