Background

Human noroviruses (HNV) are a member of the Caliciviridae family, major cause of non-bacterial gastroenteritis worldwide. HNVs cause 23 million cases of gastroenteritis, 50,000 hospitalization and 300 deaths annually in United States alone. All ages are susceptible to infection and it spreads rapidly in semi-closed communities such as hospitals, cruise ships, college campus and military bases. In infected individuals, complaints appear to be severe nausea, vomiting, and watery diarrhea within 12-24 hours, and majority of infected persons recover in 1 to 3 days. Although norovirus gastroenteritis is generally mild and self-limited short duration, infection can be severe and fatal in immune compromised and vulnerable individuals. Despite the significant economic loss and impact of public health caused by HNV, there is no efficient treatment and vaccine due to the lack of a cell culture system and small animal model.

To date, the majority of study HNV infection relies on challenging with virus to human volunteers. In experimentally infection with HNV, 11 of 16 persons showed clinical gastroenteritis (watery diarrhea and/or vomiting) with lasting of systematic illness 1-2 days and shedding virus for up to 56 days.Histological analysis of proximal intestinal biopsy specimens from ill volunteers demonstrate an intact intestinal mucosa with specific histological changes, including broadening and blunting of the villi, shortening of the microvilli, enlarged and pale mitochondria, increased cytoplasmic vacuolization, and intercellular edema. Although human volunteer studies provide materials for determine property of HNV pathogenesis, but it is still circumscribed to understand aspect of the virus. The limitations of human as a model system are i) experimentation is difficult and expensive, ii) sampling is restricted and technically demanding, iii) bio-containment requirements for the live and highly infectious virus, iv) ability to study live virus is reduced by the lack of a tissue culture system.

These difficulties associated with in vitro cultivation and human volunteer study collectively demonstrate the requirement for small animal models which is facilitated the study and lead to greater understanding of HNV.

In 2003, mouse norovirus (MNV) is discovered in immunocompromised mice lacking the recombination-activating gene 2 (RAG2) and signal transducer and activator of transcription 1 (STAT1) (RAG2/STAT1-/-). MNV l demonstrated to infect and replicate in murine macrophage cell lines and cultured primary dendritic cells and macrophages from STAT1-I- mice. Severely innate immune deficient mice were showed systemic disease and lethality with encephalitis, cerebral vasculitis, meningitis, hepatitis and pneumonia after infection. But adaptive immune system deficient mice, lacking T and B cells response, (RAG1-I- and RAG2-I- mice) are resistant to MNV 1 induced lethality. And Rag-/- mice showed high levels of viral RNA in lung, liver, spleen, proximal intestines, brain and feces while wild-type (129 and C57BL/6) cleared MNV-1 infection. These studies suggested that adaptive immunity is not necessary to protection against lethal MNV infection but are required to prevent the dissemination and continuous replication of MNV.

MNV-1 is closely related to HNV (genogroup V norovirus ― HNV is in I, II and IV) and shares numerous biochemical and genetic features, including fecal-oral transmission, replication in the intestine, and fecal shedding, with HNV [16]. And a natural host is relatively cost-effective and genetically well-characterized. These facts lead MNV to excellent model of HNV research.

MNV is most prevalent in research mice. Recent serologic study to detect antibodies against MNV in serum of laboratory mice from multiple research institutions in North America demonstrated 22.1% presence of MNV antibody in evaluated 12,639 serum samples. Furthermore 76 feces specimens collected from various breeding colonies in Berlin and Germany showed 63% of samples contained MNV RNA by one-tube real-time RT PCR method. MNV infection typically show no visible sign and clinical symptoms of disease but persist with shedding virus in feces for more than 8 weeks in immunocompetent mice [20], while MNV induce lethal infections in only severely innate immune deficient mice, such as STAT1-/- and interferonaI yR-/-. MNV is stable upon exposure to pH extremes (pH 2 to 10) and 56 degrees, remains infectious for 7 days at room temperature in fecal material. These aspects of MNV may lead to high prevalence and difficulty of eradication of MNV in mouse facilities.

This prevalence of MNV in mouse facilities may lead to unreliable and irreproducible results and impact immune response to infection with other viruses. Because both circulating macrophage and dendritic cells serve as reservoir for MNV result in inducing a robust type I IFN response in 129/SvJ mice [24] and . This issue was continually discussed and Cadwell et al reported that CR6 MNV strain, but not MNV1, induce striking abnormalities in Paneth cell function by interaction with a mutation in the Crohn's disease susceptibility gene Atgl 6L1. And Karen et al demonstrated that MNV4 alters antigen presenting activity of dendritic cells result in aggravates disease progression in a mouse model of inflammatory bowel disease [26]. In one report, MNV-G led to mouse parvovirus DNA levels higher in mesenteric lymph node, spleen, and small intestines and also virus shedding longer in BALB/c mice. These reports demonstrate that MNV infection may impact on biomedical research significantly.

Chapter 1

Mouse norovirus S7 (MNV S7) was isolated in Japan 2007 and known to most prevalent pathogen of laboratory mice. Although serologic importance of MNV S7 in mouse research colonies, pathology of MNV-S7 in mouse was not clearly understood. To assess histopathological changes from infection of MNV S7, C57BL/6, interferon gamma knockout (IFNy"/") and interferon regulator factor 3 and 7 double knockout (IRF3/74") mouse were inoculated perorally, and major organ were analyzed. Although all infected mice were seroconverted to MNV, mice showed no clinical symptoms and gross lesions except that only IRF3/7-/- mice showed mild intestinal inflammation which is related to edema, shorten vilus and activation of peyer's pathes in distal colon. Viral antigens of MNV-S7 were detected in the mesenteric lymph node of IFN7 /- and IRF3/7-/- mice, epithelium and lamina propria of IRF3/7-/- mice by immunohistochemistry. Especially, mast cells in MLN of IFN' '- were presented proliferation and matched to immunoreactive cells by probing MNV specific antibody. These data reveal possibility that MNV S7 has identical pathogenicity with MNV 1 but tropism for mast cells.

Chapter 2

Murine norovirus (MNV) is known to be the most prevalent virus in laboratory mice colonies in the world. Although it generally induces no clinical symptoms in immunocompetent mice, some studies showed that MNV alter disease progression of inflammatory bowel disease in mice. However, it has not been reported that MNV significantly impacted on another viral infection. In the present study, I examined whether or not MNV infection influence on the progression of mouse hepatitis virus (MHV) infection, which is also an important infectious disease in mice. MNV S7 and MHV-A59 were propagated using RAW 267 and DBT cells, respectively. Female, 7-weeks-old C57BL/6J mice were inoculated with 2,000 PFU of MHV-A59. A part of the mice had been inoculated with MNV-S7 per orally before one week of MHV infection. Viral growth of MHV in the liver was determined by plaque assay and the histopathological changes were examined. No significant difference was observed between mice infected MHV-A59 infection alone and those coinfected MNV-S7 and MHV A59 at 3 days postinfection. However, the growth of MHV in mice coinfected with MNV was lower than those infected MHV alone. In addition,the inflammatory lesions in the liver of mice coinfected with MNV were lighter than those infected MHV alone. IFNa production was not changed but MHV specific neutralizing antibody was amplified by previous MNV infection. This is the first report that MNV infection alters the progression of viral infection in mice.

Chapter 3

Human norovirus (HNV) cause more than 95% of gastroenteritis worldwide. Although it is realized to important in public health, there is no vaccine or drug because hampered study by the lack of a small animal model and unable culture in vitro. Various challenge of replicating HNV in vitro was unsuccessful in yielding but only mouse norovirus 1 reported that replicate in RAW 264.7 cells and has tropism for macrophage and dendritic cell. HNV infection induces gastroenteritis which is similar symptoms to food allergy as watery diarrhea due to fail of result in an imbalance in ion exchange and/or water transfer. But interaction between norovirus and mast cell, which play an important role in allergic response, was not established. The present study reports that MNV-S7 inoculated IC-2 cell (and P-815), mast cell line, showed increase of viral titer and immunofluorescent by probing MNV S7 specific antibody. These data suggest that MNV-S7 infect in mast ceIl, and aberration of mast cell is responsible for the diarrhea of MNV S7.

ノロウイルスはカリシウイルス科ノロウイルス属に属するプラス鎖single strand RNAウイルスで、ウイルス粒子は正20面体で、直径は約30nmの小さなウイルスである。5つのgenogroupに分類されており、ヒト、ウシ、ブタ、マウスからウイルスが分離、報告されている。ヒトノロウイルス(HNV)は、非細菌性急性胃腸炎の主要な原因として知られ、全世界で流行している。死に至ることはほとんど無いが、激しい下痢や嘔吐は公衆衛生上の問題となっている。まだ薬やワクチンがなく、その理由は培養細胞でHNVが増殖できないこと、およびHNV感染症の動物モデルがないためと言われている。

マウスノロウイルス(MNV)は2003年に初めて分離、報告された比較的新しいウイルスである。血清学的疫学調査で実験動物用マウスの約22%から特異抗体が検出されており、マウスの新たな病原体として注目されている。また、MNVとHNVの生化学的性状や遺伝子構成は似ており、しかもMNVは培養細胞株で増殖可能なため、MNVはHNV感染症モデルになるのではないかと期待されている。本研究では日本で分離されたMNV-S7株を用いて、MNVの性状について検討した。

第1章ではMNV-S7のマウスに対する病原性について、野生型C57BL/6マウス、interferon-γ欠損(IFN-γ-/-)マウス、interferon regulatory factor-3/7欠損(IRF-3/7-/-)マウスを用いて検討した。まず感染マウスの致死率を経口接種、脳内接種、および腹腔内接種について検討したが、死亡個体は認められなかった。次に感染マウスの病理組織学的変化とウイルス抗原の発現について検討した。その結果、MNV-S7感染野生型C57BL/6マウス、IFN-γ-/-マウスおよびIRF-3/7-/-マウスでは著明な病理組織学的変化は認められなかった。軽微な変化として、腸管粘膜固有層の水腫およびパイエル板の活性化が観察された。ウイルス抗原の発現は、感染3日後、5日後、7日後のIRF-3/7-/-マウスの腸管膜リンパ節で認められた。ウイルス抗原陽性細胞は、その形態から樹状細胞であろうと考えられた。

MNV感染が動物実験に及ぼす影響に関していろいろな報告がなされているが、概してMNV感染の影響は少ないと言われている。第2章では、マウス肝炎ウイルスA59(MHV-A59)株腹腔内接種による急性肝炎モデルに対する影響について検討した。7週齢、雌のC57BL/6マウスをMHV単独感染グループとMNVとMHVの共感染グループに分け、1週後に両方グループにMHV-A59株2,000PFUを腹腔内接種した。両グループ間の致死率には有意差は認められなかった。次に、肝炎の程度について比較した。感染3日後の両群のマウス肝臓のウイルス力価に有意差は無かったが、5日後にはMHV単独感染グループの値は共感染グループよりも有意に高かった。MHV単独感染グループの肝臓では、肝臓全体に炎症が広がっており、抗MHV抗体を用いたMHV抗原の検索では肝臓の抗原陽性細胞の割合は50%を超えていた。一方、共感染グループの肝臓の炎症は巣状であり、抗原陽性細胞の割合は25%以下だった。

両群で差が生じた原因を検討するために、血清中のIFNα量を測定したが、有意差は認められなかった。血清中のMHV中和抗体活性を測定したところ、感染5日後の共感染グループの血清中のMHV中和活性は単独感染のそれより有意に高かった。これらの結果から、MNV-S7感染マウスでは、MHV誘発肝炎の病態が軽くなることが示された。これまで、いろいろなマウスのウイルス感染症モデルに対するMNVの影響が検討されたが、ほとんど影響はないと報告されていた。本研究はMNVが他のウイルス感染症の病態を修飾することを初めて示した。

第3章ではMNV-S7の肥満細胞における増殖性について検討した。これまで、MNVはマクロファージや樹状細胞に感染すると報告されているが、繊維芽細胞などの他の細胞はMNVに対して感受性がないと報告されていた。本研究ではMNV-S7が肥満細胞で増殖するか2つの肥満細胞株を用いて検討した。IC-2細胞は未分化肥満細胞株であり、P-815細胞は分化肥満細胞株である。これらの細胞株、および陽性対照であるRAW 264細胞にMNV-S7を接種し、培養上清中のウイルス力価を経時的に測定した。その結果、MNVは2種類の肥満細胞株で増殖した。特にIC-2細胞では、RAW 264細胞と同程度によく増殖した。また、間接蛍光抗体法により感染肥満細胞株で特異蛍光が認められた。これらの結果からMNVが肥満細胞に感染することが示唆された。HNVが肥満細胞に感染するか不明であるが、この結果はHNVの発症メカニズムを考える上で非常に興味深い。

以上の結果は、MNV-S7のvirulenceは低いこと、しかしながらMHVによるウイルス性肝炎モデルの病態を軽くすること、また今まで報告されていなかった肥満細胞への感染の可能性を示した。これらの研究成果は獣医学学術上貢献するところが少なくない。よって、審査委員一同は、本論文が博士(獣医学)の学位論文として価値あるものと認めた。