Yoshinobu Kimura

Olfactory transmission of neurotropic viruses

Olfactory receptor neurons are unique in their anatomical structure and function. Each neuron is directly exposed to the external environment at the site of its dendritic nerve terminals where it is exposed to macromolecules. These molecules can be incorporated into by olfactory receptor neurons and transported transsynaptically to the central nervous system. Certain neurotropic pathogens such as herpes simplex virus and Borna disease virus make use of this physiological mechanism to invade the brain. Here the authors review the olfactory transmission of infectious agents and the resulting hazards to human and animal health.

Oral vaccination with a liposome-encapsulated influenza DNA vaccine protects mice against respiratory challenge infection.

It is well accepted that vaccination by oral administration has many advantages over injected parenteral immunization. The present study focuses on whether oral vaccination with a DNA vaccine could induce protective immunity against respiratory challenge infection. The M1 gene of influenza A virus was used to construct DNA vaccine using pcDNA 3.1(+) plasmid, a eukaryotic expression vector. The cationic liposomes were used to deliver the constructed DNA vaccine. In vitro and in vivo expression of M1 gene was observed in the cell line and in the intestine of orally vaccinated C57BL/6 mice, respectively. It became clear that this type of oral DNA vaccination was capable of inducing both humoral and cellular immune responses, together with an augmentation of IFN‐γ production. In addition, oral vaccination with liposome‐encapsulated DNA vaccine could protect the mice against respiratory challenge infection. These results suggest that gastrointestinal tract, a constituent member of the common mucosal immune system, is a potent candidate applicable as a DNA vaccine route against virus respiratory diseases. J. Med. Virol. 86:886–894, 2014.

The US3 protein kinase of herpes simplex virus attenuates the activation of the c-Jun N-terminal protein kinase signal transduction pathway in infected piriform cortex neurons of C57BL/6 mice

Stereotaxic microinjection of herpes simplex virus (HSV) into the mouse olfactory bulb resulted in infection of neurons of the piriform cortex. Neurons infected with the wildtype HSV showed no evident phosphorylation of c-Jun N-terminal protein kinase (JNK)/c-Jun. In contrast, neurons infected with a US3 gene-disrupted mutant of the L1BR1 virus displayed phosphorylated JNK/c-Jun in a nuclear staining fashion. Induction of neuronal apoptosis by the wildtype HSV was partially suppressed when compared with that of the L1BR1 virus. A US3-rescued isolate of the L1B(-)11 virus behaved as did the wildtype virus. Collectively, the US3 protein kinase of HSV plays a role in attenuating the virus-induced activation of the JNK signal transduction pathway in the central nervous system and may contribute, at least in part, to controlling neuronal apoptosis.

Respiratory syncytial virus protects against the subsequent development of ovalbumin‐induced allergic responses by inhibiting Th2‐type γδ T cells

Respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans still remain inconclusive. The association between RSV infection and allergic diseases may be dependent on an atopic background and previous history of RSV infection. It has been reported that RSV infection before sensitization to an allergen decreased the production of Th2‐like cytokines in the lung and the levels of allergen‐specific Th2‐type antibodies in the serum. However, the underlying mechanisms are largely unknown. In the present study, the role of pulmonary γδ T cells in RSV‐affected, allergen‐induced airway inflammation was investigated. BALB/c mice were sensitized to or challenged with ovalbumin (OVA) and infected with RSV either before or after the sensitization period. It became clear that sensitization and challenge of mice with OVA induced a large influx of γδ T cells to the lungs. However, prior RSV infection inhibited the infiltration of γδ T cells as well as activated γδ T cells, characterized by expression of CD40L or CD69 molecular in the cell surface. Moreover, prior RSV infection elevated the type 1 cytokine gene expression but suppressed type 2 cytokine expression in the lung γδ T cells. Adoptive transfer of γδ T cells from OVA‐sensitized and challenged mice increased airway inflammation, suggesting that γδ T cells may play a proinflammatory role in allergic responses. These results described here support the idea of an unknown γδ T cell‐dependent mechanism in the regulation of RSV‐affected, allergen‐induced allergic airway responses. J. Med. Virol. 85:149–156, 2012.

Fetal calf serum inhibits virus genome expression in Madin-Darby canine kidney cells persistently infected with influenza A virus

A cell line of Madin-Darby canine kidney (MDCK) cells persistently infected with human influenza A virus has been established and designated as MDCK-IVpi cells. Production of progeny virus in MDCK-IVpi cells was suppressed when the cells were incubated in the presence of 10% fetal calf serum (FCS). FCS impaired virus mRNA synthesis in MDCK-IVpi cells, which resulted in a scarcity of virus proteins for virion formation. However, MDCK-IVpi cells well supported the growth of superinfecting heterologous influenza viruses, even in the presence of FCS. A certain fetuin-like substance in FCS might be responsible for the observed inhibition of virus replication.

PCR Search for the Herpes Simplex Virus Type 1 Genome in Brain Sections of Patients with Familial Alzheimer's Disease

An association between Chlamydia pneumoniae and Alzheimers disease (AD) remains questionable (3, 5). Herpes simplex virus type 1 (HSV-1), the most ubiquitous neurotropic virus in humans, has been proposed as a risk factor for AD, although this issue is still under debate. Itzhaki et al. raised the possibility that the presence of HSV-1 in the brain is a risk factor for AD in elderly people carrying the apolipoprotein E ɛ4 allele (4). Strandberg noticed a significant association between serologically measured viral burden and cognitive impairment in elderly people (6). On the other hand, Beffert et al. argued that HSV-1 in the brain does not confer an increased risk for AD when combined with the apolipoprotein E ɛ4 allele and that ɛ4 carriers are not more susceptible to HSV-1 infection than are non-ɛ4 carriers (1). n nFamilial AD, the least common type of AD, is characterized by the early onset of the disease (at 35 to 60 years of age) with early neuropathological alterations, including β-amyloid deposition. Mutations on the amyloid precursor protein, presenilin-1, and presenilin-2 genes have been identified in familial AD patients. However, other possible mechanisms underlying the pathology of familial AD, including the involvement of HSV-1 in the deterioration phase of the disease, have not been addressed. n nBy using nested PCR, we examined formalin-fixed, paraffin-embedded, postmortem brain tissue from three individuals with familial AD, six without AD, and two with sporadic AD for the presence of HSV-1 glycoprotein D (Table u200b(Table1).1). A mutation on the presenilin-1 gene had been identified in patient 1. HSV-1 DNA was detected in the brains of all three familial-AD individuals, preferentially in the frontal and temporal lobes, whereas it was not found in any parts of the brains of non-AD group members except patient 9, whose frontal lobe was HSV-1 positive. In situ hybridization (using a DAKO GenPoint tyramide signal amplification system and an ENZO HSV DNA probe) detected HSV DNA in the frontal and temporal cortex neurons of patients 1 and 2, with the virus-specific signal being detected predominantly in the cytoplasm in a dotlike staining pattern. Furthermore, high-sensitivity immunohistochemistry (DAKO ENVISION+) detected HSV-1 antigens in the frontal- and temporal-cortex neurons of patients 1 and 2 in a cytoplasmic staining fashion. A moderate to high level of β-amyloid deposition was detected in the brains of all three familial- and both sporadic-AD patients, whereas it was not evident in the non-AD brains except that of patient 9, whose brain exhibited moderate β-amyloid deposition. n n n nTABLE 1. n nDetection of the HSV-1 genome in brain sections of patients with familial AD n n n nThese results (the presence of viral DNA and antigens in neuronal cytoplasm) are suggestive of limited reactivation of HSV-1, coupled with β-amyloid deposition, in the brains of individuals with familial AD. In a latent state, virus genomes are harbored in the nucleus but not in the cytoplasm of neurons (7). Significantly, β-amyloid fibrils have been demonstrated to stimulate infection with enveloped viruses, including HSV, in vitro (8). Additionally, glycoprotein B of HSV-1, the internal sequence of which has homology to the carboxyl-terminal region of the β-amyloid peptide, has been shown to promote fibril formation in vitro (2). Thus, there appears to exist a close association between β-amyloid deposition and HSV-1 reactivation in the human brain. Since repeated reactivation of HSV-1 in neurons likely promotes neurodegeneration in AD brains, preventive and therapeutic measures against HSV-1 may be applied not only to sporadic but also to familial AD.

Evaluation of a Virus Derived From MDCK Cells Infected Persistently With Influenza A Virus as a Potential Live-Attenuated Vaccine Candidate in the Mouse Model

A temperature‐sensitive mutant virus unable to replicate at 38°C was recovered from passage 189 (IVpi‐189) of Madin‐Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi‐189 virus‐infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non‐permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61‐24‐P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi‐189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi‐189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi‐189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi‐189 virus, an avirulent temperature‐sensitive mutant, is a promising candidate for use as a live‐attenuated vaccine. J. Med. Virol. 80:888–894, 2008.

A Prophylactic Effect of an Oligodeoxynucleotide Containing a Cytidine–Guanosine Motif against Japanese Cedar Pollen-Induced T-Helper Type 2 Allergic Response

Background. Over 10% of entire population in Japan suffer from allergic diseases induced by Japanese cedar pollen (JCP) every spring. In terms of preventive medicine, it has become a matter of urgency to establish successful prophylactic and therapeutic strategies for controlling the disorders. The effect of an oligodeoxynucleotide containing a cytidine–guanosine motif (CpG ODN) on the regulation of immune responses induced by JCP was investigated in this study. Methods. BALB/c mice were inoculated with CpG ODN intraperitoneally before intranasal sensitization to JCP. Cellular infiltration in the lung of BALB/c mice after treatment with CpG ODN or JCP was performed by hematoxylin and eosin (H&E) staining. Antibody titers and cytokines levels were determined by ELISA. Results. Intranasal inoculation of BALB/c mice with JCP induced a T-helper type 2 (Th2-type) dominant immune response, as characterized by the production of interleukin (IL)-4 and IL-5 in the lung and of JCP-specific IgE antibody in serum. Prior intraperitoneal administration of CpG ODN to mice suppressed the subsequent JCP-induced antibody production and infiltration of inflammatory cells in the lung. The inhibitory mechanism of CpG ODN seemed to be attributable to a CpG ODN-induced Th1-type dominant environment, which down-regulated Th2-type response subsequently induced by JCP allergen sensitization. Furthermore, administration with CpG ODN decreased the production of JCP-induced IL-17, which has been found to play a pivotal role in several inflammatory diseases including allergic asthma. The decreased production of IL-17, together with reduced secretion of IL-4 and IL-5, may contribute to diminish the inflammation in the lung of JCP-sensitized mice. Conclusion. This work provides evidence that the CpG ODN has a prophylactic effect on the JCP-induced Th2-type allergic responses by establishing or restoring a Th1-type shift of immune environments.

Infection with respiratory syncytial virus influences FasL-mediated apoptosis of pulmonary γδ T cells in a murine model of allergen sensitization.

Abstract Background: It has been reported that adoptive transfer of γδ T cells increases the cellular infiltration, especially eosinophils, in the lungs of allergic mice, suggesting that γδ T cells may play a proinflammatory role in allergic airway inflammation. Respiratory syncytial virus (RSV) infection can decrease the number of Th2-type γδ T cells. However, the underlying mechanisms remain unknown. Methods: BALB/c mice were inoculated intranasally with RSV before or after sensitization to OVA. The amounts of Th1/Th2 cytokines as well as the levels of specific antibodies were determined by ELISA. The apoptotic death of pulmonary γδ T cells was analyzed by flow cytometry. Results: Adoptive transfer of γδ T cells increased the production of Th2 cytokines in the lungs and allergy-related antibodies in the serum, further confirming that γδ T cells act as pro-inflammatory cells or a promoter for the development of allergic asthma. RSV infection before sensitization to OVA enhanced apoptotic death of pulmonary γδ T cells. The percentage and absolute number of FasL-expressing γδ T cells in the lungs of allergic mice were elicited significantly by prior RSV infection. Blocking FasL with monoclonal antibody diminished apoptotic death of γδ T cells, suggesting that FasL is important for RSV-induced apoptosis of pulmonary γδ T cells. Conclusions: This work provides evidence that RSV infection suppresses the subsequent development of OVA-induced allergic responses partly by enhancing FasL-mediated apoptosis of pulmonary γδ T cells.