Yukiko Tomioka

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow.

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.

Characterization of novel avian paramyxovirus strain APMV/Shimane67 isolated from migratory wild geese in Japan.

An avian paramyxovirus (APMV) isolated from goose feces (APMV/Shimane67) was biologically, serologically and genetically characterized. APMV/Shimane67 showed typical paramyxovirus morphology on electron microscopy. On hemagglutination inhibition test, antiserum against APMV/Shimane67 revealed low reactivity with other APMV serotypes and vice versa. The fusion (F) protein gene of APMV/Shimane67 contained 1,638 nucleotides in a single open reading frame encoding a protein of 545 amino acids. The cleavage site of F protein contained a pair of single basic amino acid (VRENR/L). The nucleotide and deduced amino acid sequences of the F gene of APMV/Shimane67 had relatively low identities (42.9–62.7% and 28.9–67.3%, respectively) with those of other APMVs. Phylogenetic analysis showed that APMV/Shimane67 was related to NDV, APMV-9 and APMV-12, but was distinct from those APMV serotypes. These results suggest that APMV/Shimane67 is a new APMV serotype, APMV-13.

In ovo infection with an avian leukosis virus causing fowl glioma: viral distribution and pathogenesis.

We have previously isolated an avian leukosis virus (ALV) from a chicken affected with so-called fowl glioma. A resistance-inducing factor test indicated that the isolate was classified into a subgroup A. The distribution and pathogenicity were investigated in C/O specific pathogen free chickens infected in ovo with this virus. Histologically, 11 of 12 (92%) infected birds had non-suppurative encephalitis and three birds (25%) showed the characteristic nodules of fowl glioma at 50 or 100 days of age. Non-suppurative myocarditis with matrix inclusions and atypical myocytes were also noted in nine (75%) of the birds and the ALV antigens were immunohistochemically detected in various general organs as well as the central nervous system and heart. The semi-quantitative determination of the proviral DNA and viral RNA supported the immunohistochemical results and indicated that the virus was likely to replicate especially in myocardial fibres. The isolated ALV failed to induce other neoplastic lesions in this line of chickens within the experimental period of 100 days, despite the broad tissue tropism throughout the body. These results confirmed that this virus was able to induce glioma in embryo-inoculated chickens.

Valine 1532 of human BRC repeat 4 plays an important role in the interaction between BRCA2 and RAD51

hBRC4 binds to hRAD51 by enzyme linked immunosorbent assay (View interaction)

Multiple Perineuriomas in Chicken (Gallus gallus domesticus)

Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100α/β protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice.

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

Cerebellar pathology in transgenic mice expressing the pseudorabies virus immediate‐early protein IE180

Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate‐early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl‐terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.

The first immunoglobulin-like domain of porcine nectin-1 is sufficient to confer resistance to pseudorabies virus infection in transgenic mice

Summary.Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). Porcine nectin-1 mediates entry of pseudorabies virus (PRV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and bovine herpesvirus type 1 (BHV-1). The gD-binding domain of nectin-1 is the first or N-terminal immunoglobulin (Ig)-like domain of the entire ectodomain. Here, we generated three transgenic mouse lines expressing a fusion protein consisting of the first Ig-like domain of porcine nectin-1 and the Fc portion of porcine IgG1 to assess the antiviral potential of the first Ig-like domain of nectin-1 in vivo. All of the transgenic mouse lines showed significant resistance to PRV infection via intraperitoneal inoculation (survival rates of 67% to 100%). In the intranasal challenge, a lower but still significant protection was observed; 21% to 55% of the animals from the three transgenic mouse lines survived. The present results demonstrate that a soluble form of the first domain of porcine nectin-1 is able to exert a significant antiviral effect against pseudorabies virus infection.

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.

Fusion protein consisting of the first immunoglobulin‐like domain of porcine nectin‐1 and Fc portion of human IgG1 provides a marked resistance against pseudorabies virus infection to transgenic mice

Nectin‐1 is a Ca2+‐independent Ig‐like cell–cell adhesion molecule and an alphaherpesvirus receptor that binds to virion glycoprotein D by the first Ig‐like domain. We have investigated the antiviral potentials of soluble forms of porcine nectin‐1 to PRV infection by generating transgenic mice expressing different types of fusion protein. Previously, we reported that mice transgenic for a chimera that carried the entire ectodomain of porcine nectin‐1 fused to the Fc portion of porcine IgG1 were more resistant than those transgenic for a chimera that carried the first Ig‐like domain fused to the Fc portion. Recently, we generated transgenic mice expressing a fusion protein made of the first Ig‐like domain fused to the Fc portion of human IgG1, and reported that they showed a microphthalmia. Here, two transgenic mouse lines expressing the fusion protein were challenged with PRV for comparing their resistances with those of transgenic mice expressing different types of fusion protein. Surprisingly, both transgenic mouse lines showed a high resistance to the viral infection, especially via the i.n. route. Significant resistance of the embryonic fibroblasts was also observed. Altogether, these findings indicated that the fusion protein consisting of the first Ig‐like domain fused to the human Fc portion provided a marked resistance against PRV infection to the transgenic mice.