Lela K. Riley

Progress and prospects in rat genetics: a community view

The rat is an important system for modeling human disease. Four years ago, the rich 150-year history of rat research was transformed by the sequencing of the rat genome, ushering in an era of exceptional opportunity for identifying genes and pathways underlying disease phenotypes. Genome-wide association studies in human populations have recently provided a direct approach for finding robust genetic associations in common diseases, but identifying the precise genes and their mechanisms of action remains problematic. In the context of significant progress in rat genomic resources over the past decade, we outline achievements in rat gene discovery to date, show how these findings have been translated to human disease, and document an increasing pace of discovery of new disease genes, pathways and mechanisms. Finally, we present a set of principles that justify continuing and strengthening genetic studies in the rat model, and further development of genomic infrastructure for rat research.

Development of a Microsphere-Based Serologic Multiplexed Fluorescent Immunoassay and a Reverse Transcriptase PCR Assay To Detect Murine Norovirus 1 Infection in Mice

ABSTRACT Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n = 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection.

Diagnostic testing of mouse and rat colonies for infectious agents.

Rodent health monitoring programs make an essential contribution to biomedical research by identifying the presence of infectious agents that might confound animal research. The authors discuss the types of diagnostic tests available, which agents deserve monitoring, and the appropriate frequency for such interventions.

Completion of the swine genome will simplify the production of swine as a large animal biomedical model

BackgroundAnatomic and physiological similarities to the human make swine an excellent large animal model for human health and disease.MethodsCloning from a modified somatic cell, which can be determined in cells prior to making the animal, is the only method available for the production of targeted modifications in swine.ResultsSince some strains of swine are similar in size to humans, technologies that have been developed for swine can be readily adapted to humans and vice versa. Here the importance of swine as a biomedical model, current technologies to produce genetically enhanced swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model are discussed.ConclusionsThe completion of the swine genome will enhance the continued use and development of swine as models of human health, syndromes and conditions.

Molecular characterization of three novel murine noroviruses

Murine noroviruses (MNV) comprise a group of newly recognized pathogens infecting laboratory mice. The first reported murine norovirus, murine norovirus 1 (MNV-1), produces a transient infection with a short duration of fecal shedding after infection of immunocompetent laboratory mice. Our laboratory subsequently isolated three novel murine noroviruses, murine norovirus 2 (MNV-2), murine norovirus 3 (MNV-3), and murine norovirus 4 (MNV-4), that have markedly different pathogenicity from MNV-1 by producing persistent infections and prolonged fecal shedding in infected immunocompetent mice. In this study, the nucleotide sequences and the predicted amino acid sequences of the three novel murine noroviruses were determined and compared to each other, MNV-1, and other previously described human and animal noroviruses. The three novel murine norovirus strains were shown to be related to each other and MNV-1 by sequence and phylogenetic analysis even though MNV-2, MNV-3 and MNV-4 all display markedly different biologic behavior from that of MNV-1.

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species.

ABSTRACT Over the past decade, several Helicobacter species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents. In this report, we describe a novel urease-negative helicobacter,Helicobacter typhlonius sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections. H. typhlonius is genetically most closely related to H. hepaticus. Like H. hepaticus, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H. hepaticus. Notably, H. typhlonius does not produce urease or H2S nor does it hydrolize indoxyl-acetate. Compared to other Helicobacter species that commonly colonize rodents,H. typhlonius was found to be less prevalent than H. hepaticus and H. rodentium but as prevalent asH. bilis. H. typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

Molecular characterization of newly recognized rodent parvoviruses

Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).

Serodiagnosis of Mice Minute Virus and Mouse Parvovirus Infections in Mice by Enzyme-Linked Immunosorbent Assay with Baculovirus-Expressed Recombinant VP2 Proteins

ABSTRACT Mice minute virus (MMV) and mouse parvovirus (MPV) type 1 are the two parvoviruses known to naturally infect laboratory mice and are among the most prevalent infectious agents found in contemporary laboratory mouse colonies. Serologic assays are commonly used to diagnose MMV and MPV infections in laboratory mice; however, highly accurate, high-throughput serologic assays for the detection of MMV- and MPV-infected mice are needed. To this end, the major capsid viral protein (VP2) genes of MMV and MPV were cloned and MMV recombinant VP2 (rVP2) and MPV rVP2 proteins were expressed by using a baculovirus system. MMV rVP2 and MPV rVP2 spontaneously formed virus-like particles that were morphologically similar to empty parvovirus capsids. These proteins were used as antigens in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MMV or anti-MPV antibodies in the sera of infected mice. Sera from mice experimentally infected with MMV (n = 43) or MPV (n = 35) and sera from uninfected mice (n = 30) were used to evaluate the ELISAs. The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice, and the MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice. Both assays outperformed a parvovirus ELISA that uses a recombinant nonstructural protein (NS1) of MMV as antigen. The MMV rVP2 and MPV rVP2 proteins provide a ready source of easily produced antigen, and the ELISAs developed provide highly accurate, high-throughput assays for the serodiagnosis of MMV and MPV infections in laboratory mice.

Molecular characterization of three newly recognized rat parvoviruses

Rodent parvoviruses have been documented to interfere with both in vivo and in vitro research. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.

Detecting Food- and Waterborne Viruses by Surface-Enhanced Raman Spectroscopy

Abstract:  Food‐ and waterborne viruses pose serious health risks to humans and were associated with many outbreaks worldwide. Rapid, accurate, and nondestructive methods for detection of viruses are of great importance to protect public health. In this study, surface‐enhanced Raman spectroscopy (SERS) coupled with gold SERS‐active substrates was used to detect and discriminate 7 food‐ and waterborne viruses, including norovirus, adenovirus, parvovirus, rotavirus, coronavirus, paramyxovirus, and herpersvirus. Virus samples were purified and dialyzed in phosphate buffered saline (8 to 9 log PFU/mL) and then further diluted in deionized water for SERS measurement. After capturing the characteristic SERS spectral patterns, multivariate statistical analyses, including soft independent modeling of class analogy (SIMCA) and principal component analysis (PCA), were employed to analyze SERS spectral data for characterization and identification of viruses. The results show that SIMCA was able to differentiate viruses with and without envelope with >95% of classification accuracy, while PCA presented clear spectral data segregations between different virus strains. The virus detection limit by SERS using gold substrates reached a titer of 102. Practical Application:  SERS is a simple, rapid, and accurate method for detection of food‐ and waterborne viruses. Our results demonstrate that coupled with gold substrates, SERS was able to rapidly detect and discriminate among different food‐ and waterborne viruses, indicating that SERS can provide rapid, sensitive, and reproducible detection results with minimum sample preparation for virus detection.