In 2003 there was one published report by Karst et al. (1) of a calicivirus (non-enveloped, single-stranded, RNA virus) isolated from a colony of immune deficient mice experiencing unexpected mortality. These mice (RAG/STAT-/-) were deficient in recombination-activating gene 2 (RAG) and in signal transducer and activator of transcription 1 (STAT1). Subsequently, the authors described the virus isolated from these animals as a norovirus, naming it Murine Norovirus 1 (MNV-1). The authors describe the requirement of STAT1-dependent innate immunity for resistance to the murine norovirus. These cytoplasmic proteins, or STATs, are activated by ligand-activated tyrosine kinases via their phosphorylation of a single STAT Tyr residue. The phosphorylated STAT dimerizes, enters the nucleus, binds to DNA and stimulates the transcription of specific genes(2). The authors reported that mice lacking both interferon αβ and interferon γ receptors were very susceptible to lethal infection demonstrating that interferons are needed for resistance to MNV-1. STAT1 is involved in signaling through both IFN αβ and IFNγ receptors. RAG/STAT-/- mice inoculated with gradient purified virus showed encephalitis, cerebral vasculitis, pneumonia, meningitis and hepatitis (1).
Immunocompetent mice (129 wild-type) seroconverted to MNV-1 capsid protein after oral or intracerebral inoculation but had no clinical signs or tissue pathology and a transient infection as evidenced by lack of viral RNA from tissues by 3 days post-innoculation (1). This type of clearance was consistent with clearance by innate immunity. In mice deficient in T and B lymphocytes, MNV-1 was found to cause persistent infection but no clinical signs were reported(1).
This publication was the first report of a norovirus of murine origin.
Since the initial report in 2003, more information concerning MNV-1 has been elucidated. In 2004, Wobus et al. (3) developed a cell-culture based system for Murine Norovirus 1. This was the first report of successful in vitro cultivation of a norovirus as attempts with human norovirus have previously failed. During infectivity studies, Wobus and colleagues examined tissues from STAT1-/- mice orally infected with MNV-1 by immunohistochemistry. By Day 2 post inoculation, positive staining cells were identified in the spleen and liver. The cells identified as positive for viral particles included Kupffer cells in the liver and macrophages and dendritic cells in the spleen. When examined in vitro, it was found that both macrophages and dendritic cells support the growth of the virus in culture. Both wild type and STAT1-/- cells supported viral propagation with the latter yielding higher titers. Cells lacking the IFNαβγR or IFNαβR also supported viral growth. These findings continue to underline the importance of the innate immune response to this virus. Another important finding from this group was that serial passage of the virus in cell culture caused viral attenuation. This appeared to be the result of changes in the capsid protein.
During the 56th AALAS National Meeting held in St.Louis, Missouri(4), Dr. H. Virgin reported that RAG-/- mice did not experience lethal infection when inoculated with MNV 1, and demonstrated no dramatic pathology although they were persistently infected with virus still detectable 90 days post-inoculation. Thus, the virus was persistently shed and maintained in the immunocompromised animals. He postulated that spread of the virus was fecal-oral as dirty bedding could be used to infect naïve animals. A fecal-oral transmission route has been reported for the transfer of human noroviruses as well. In addition, seroconversion was reported to occur 28-35 days post-inoculation. Dr. Virgin also reported that, as in human norovirus, there appeared to be several mouse isolates of norovirus that were closely related, differing by only a few amino acids.
On a practical note he re-emphasized the finding that a 1:10 dilution of bleach is the only disinfectant that is currently known to kill the virus, although autoclaving of materials is also effective. This has also been described elsewhere for human noroviruses(9) with quaternary ammonium compounds being described as ineffective. Rederivation is an effective means for exclusion of Mouse Norovirus-1 (4) from susceptible populations.
Although noroviruses are endemic in the human population, the pathophysiology is currently unknown and no known animal reservoir has been identified. The information Dr. Virgin’s laboratory has generated will most likely be extremely useful in future studies for human norovirus.
At an informational seminar held in Boston, MA, in October of 2005 (5), results presented from a natural infection study in CD-1 mice showed there was poor to no seroconversion at three (3) weeks post-infection, the time normally cited as the minimum length of time for antibody presence in sera. Animals maintained on dirty bedding also showed a poor antibody response at 4 weeks post exposure that was improved at 5-6 weeks. These findings thus point to the need for a longer period of time before detectable antibodies are present in the serum as compared to the more well known viruses. In addition, given the persistence of the virus in the cage environment and in an infected host, PCR could be used on feces and intestine as soon as Day 1-2 post-infection up to a period in excess of 6 weeks.
In October of 2005, Hsu et al. (6) described the detection of MNV-1 using a microsphere-based serologic multiplexed fluorescent immunoassay. The prevalence of the virus based on submitted serum samples that tested positive for antibodies to MNV-1 was reported as 22.1%. In contrast, the same laboratory reports a prevalence of 2.5% for mouse parvovirus. Thus it appears that many facilities are contaminated with MNV-1. This would not be unexpected, as the authors point out, this virus is newly recognized, cannot be diagnosed based on clinical signs and most institutions are just now beginning to put practices and procedures into place to control or eliminate the virus from their colonies. Hsu and colleagues also reported (6) that, using PCR, MNV-1 was detected in feces of ICR mice out to at least Day 7 post inoculation by oral gavage. At 5 weeks, fecal PCR was negative. However, at 5 weeks post inoculation virus was present by PCR in the spleen, jejunum and mesenteric lymph node. This data was in contrast to the original data of Karst et al. (1) using 129 mice.
At the 56th AALAS National Meeting in St. Louis, Missouri, Hsu et al. (7) presented data demonstrating the presence of additional isotypes of MNV that they called MNV-2, MNV-3 and MNV-4. These isotypes had 86-91% nucleotide identity with some geographic distribution. Cross-reactivity was seen between these isotypes in the laboratory’s serologic testing. It is not unexpected that additional isotypes were identified and is highly likely that even more will be recognized as this is what occurs for human norovirus (8).
New data from the laboratory (7) also demonstrated that PCR could be used to detect MNV-2, -3 and -4 from the spleen, jejunum and mesenteric lymph nodes even at 8 weeks post infection. Jejunum and mesenteric lymph nodes were the best tissues to select. Even fecal shedding was still occurring for MNV-2, -3 and -4 at Week 8. This is unlike what was previously reported for MNV-1 highlighting possible diagnostic as well as control issues that may be dependent on the isotype present. These types of issues have already caused difficulties in human diagnostics as it has been reported that the considerable variability, genetically and antigenically, of human noroviruses has made it difficult to develop assays that are equally sensitive against all the variants (9). This has lead to a false negative problem with low sensitivity of PCR and antigen capture assays.
It is anticipated that additional information concerning Murine Nororvirus and its potential role in the study of human nororviruses will continue to be published. It will also be of interest to researchers, veterinarians and facility managers how this virus may potentially impact the health and utility of the many rodent models currently in use. To date the only published report of animal health concerns is the original paper by Karst et al. in 2003 (1).
Just as Murine Norovirus will potentially become a useful model for the human noroviruses, the human virus may foretell what is to come for MNV. The genus Norovirus is in the family Caliciviridae which also includes the genera Vesivirus, Sapovirus, and Lagovirus. In the United States noroviruses are a major cause of viral gastroenteritis in humans (8). Some individuals can develop chronic disease however most cases are acute disease of 1-2 days duration.
Several studies have shown that for noroviruses there is within genogroup recombination that gives rise to hybrid strains capable of causing outbreaks in humans. In 2005 (8) 124 Norovirus sequences had been recognized in humans. Once new Noroviruses form they spread rapidly around the world. Etherington et al. (8) also reported that the genome of the virus appears to have a "hotspot" for recombination located near the end of the open reading frame 1 (non-structural polyprotein) and the start of open reading frame 2 (capsid protein). As discussed previously, new strains of murine norovirus are also emerging.
In a review by Radford et al. published in 2004 (9), noroviruses are described as having the potential to cause significant morbidity. This is evident as many reports of outbreaks on cruise ships, in hospitals or other institutions have made the headline news. Noroviruses are also described as being capable of causing mortality in immunocompromised individuals or in individuals living in developing countries. Some patients also show resistance, the mechanism of which is currently unknown. Resistance is postulated to be due to the viruses binding affinity for different antigens. Again there are obvious similarities with what is currently being reported in the mouse models examined with lethal disease in some immunodeficient models. Of potential relevance to maintaining mouse colonies is that immunosuppressed humans have been shown to shed virus for many months (9).
Currently there is no evidence for zoonotic transmission of the norovirus.
In human diagnostics, RT-PCR is described as the gold standard for norovirus diagnostics (9). However, due to the considerable variability of the human noroviruses, it has been difficult to develop assays that are equally sensitive against all of the variants. Thus, Radford et al. describe false negatives as being a problem due to the low sensitivity of existing RT-PCR assays (9). This has also been an issue with antigen capture assays. The probability of these issues arising in the diagnosis of murine norovirus is also very high. In fact, at the October 2005 seminar held in Boston (5), it was presented that several reiterations of an RT-PCR assay had to be developed to find one that would detect all currently recognized isolates of MNV. Thus, it will be a diagnostic challenge to update assays for MNV as new isolates are discovered.
In the Spring of 2005, Taconic tested all of its commercial US colonies for the presence of MNV and all results were negative. Since that testing, a meeting convened in 2005 between all Taconic veterinarians and PhD scientists made the decision to add testing for MNV to the IHMS52 diagnostic panel currently utilized in all US colony health testing. Customers of Taconic Anmed purchasing an IHMS52 diagnostic panel will also receive this testing.
Taconic is confident that its current production methodology is designed to prevent any pathogenic agents, such as MNV, from gaining access to the colonies. All animals that are intended for production at facilities must be either caesarean section or embryo transfer derived onto germ free or defined flora stock before entry onto the Production site. After the derivation, the microbial flora is monitored at weekly intervals and the recipient females and derived offspring are tested, as well, for all agents on our health monitoring program. After successful derivation testing the animals are associated with a cocktail of 8 anaerobic bacteria (Altered Schaedler Flora) to normalize the gut and prevent GI changes associated with germ free animals (cecal dilation). All of the commercial models have been derived in this manner prior to production of animals for sale. Outbred models are then transferred to barriers while inbred and congenic lines are bred in pyramid colony management plan. Foundation colony animals are reared in a gnotobiotic fashion with strict monitoring and attention to microbial flora as the greatest risk of contamination is with environmental bacteria, yeasts and fungi.
Breeder animals are transferred to Taconic’s Isolated Barrier Units from the gnotobiotic foundation colonies. All materials entering the barrier are sterilized by steam sterilization, ethylene oxide, cold sterilants or gamma irradiation. Strictly controlled procedures are in effect for entry of personnel into the barriers. Taconic also has very stringent policies on pets and personnel movement to minimize the risk of contamination to our barriers.
Taconic is fully committed to the highest health standards utilizing the most current methodologies and has a track record of over 20 years of continual high health quality.
Taconic Anmed’s Molecular Diagnostics laboratory, led by Dr. Heinz Reiske, has been developing a RT-PCR assay for Murine Norovirus. The advantage of PCR is its ability to detect minute amounts of MNV rapidly, especially before the on-set of seroconversion in infected animals. In addition to its utility for clinical specimens, PCR can be run on swabs or fecal pellets recovered from the cage providing an assay useful in environmental monitoring. Given the genetic diversity of noroviruses in general, and MNV in particular, Taconic Anmed is sequencing field isolates to insure optimum diagnostic sensitivity of molecular assays for MNV.
Serological testing compliments PCR testing, and is especially useful for detecting agents with a high degree of genetic diversity. Thus, to provide our clients with the most comprehensive testing regimen, Taconic Anmed, through efforts lead by Dr. Fatima Jones, is developing immunoassays for MNV. Both testing methods are under development and will be available to customers in 2006.
The veterinary and scientific staff at Taconic is available to discuss any questions, concerns or issues you may have surrounding Murine Norovirus. You can also contact your Taconic Area Technical Representative or Taconic Customer Service for additional information.
