Chun-Ho Park

The invasion routes of neurovirulent A/Hong Kong/483/97 (H5N1) influenza virus into the central nervous system after respiratory infection in mice

Summary.A/Hong Kong/483/97 (H5N1) influenza virus (HK483) isolated from the third patient during the outbreak of chicken and human influenza in Hong Kong in 1997 was shown to be neurovirulent in mice. HK483 was inoculated intranasally to mice, and the invasion routes of the virus in the central nervous system (CNS) were investigated by immunohistochemical and in situ hybridization. The pathological changes consisted of bronchopneumonia, ganglionitis, and nonpurulent encephalomyelitis of the brain stem and the anterior part of the thoracic cord. Viral antigens and viral nucleic acids (RNA and mRNA) were demonstrated in the pterygopalatine, trigeminal and superior ganglions prior to or simultaneously with their detection in the CNS. The antigens and nucleic acids were also observed in the olfactory bulb from an early stage of the infection. In the spinal cord, virus-infected cells were first demonstrated in the grey matter of the thoracic cord. The virus, which primarily replicated in the lungs, was considered to invade the thoracic cord via cardiopulmonary splanchnic nerves and sympathetic nerves. These findings indicate that the virus reached the CNS through afferent fibers of the olfactory, vagal, trigeminal, and sympathetic nerves following replication in the respiratory mucosa.

Neurotropism of the 1997 Hong Kong H5N1 influenza virus in mice

The direct transmission of H5N1 influenza A viruses from chickens to humans in Hong Kong in 1997 emphasized the need to have information on the pathogenesis of avian influenza virus infection in mammals. H5N1 influenza viruses isolated from patients during the incident killed experimentally infected mice. The principal lesions of the mice were broncho-interstitial pneumonia and nonsuppurative encephalitis. Infectious viruses and/or viral antigens were detected in the brain as well as in the trigeminal and vagal ganglia but not in the blood of the mice. These findings suggest that the virus reached the brain through the vagus and/or trigeminal nerves following replication in the respiratory mucosa. The results imply that neurotropism of the H5N1 virus in mice is a novel characteristic in the pathogenesis of infection by human influenza virus isolates.

Serological evidence of transmission of human influenza A and B viruses to Caspian seals (Phoca caspica)

Seroepidemiological surveillance of influenza in Caspian seals (Phoca caspica) was conducted. Antibodies to influenza A virus were detected in 54% (7/13), 57% (4/7), 40% (6/15) and 26% (11/42) of the serum samples collected in 1993, 1997, 1998 and 2000 by enzyme‐linked immunosorbent assay (ELISA). In an hemagglutination‐inhibition (HI) test using H1‐H15 reference influenza A viruses as antigens, more than half of the examined ELISA‐positive sera reacted with an H3N2 prototype strain A/Aichi/2/68. These sera were then examined by HI test with a series of naturally occurring antigenic variants of human H3N2 virus, and H3 viruses of swine, duck, and equine origin. The sera reacted strongly with the A/Bangkok/1/79 (H3N2) strain, which was prevalent in humans in 1979–1981. The present results indicate that human A/Bangkok/1/79‐like virus was transmitted to Caspian seals probably in the early 1980s, and was circulated in the population. Antibodies to influenza B virus were detected by ELISA in 14% (1/7) and 10% (4/42) serum samples collected from Caspian seals in 1997 and 2000, respectively. Our findings indicate that seal might be a reservoir of both influenza A and B viruses originated from humans.

Highly Pathogenic H5N1 Influenza Virus Causes Coagulopathy in Chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus‐infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.

The Vagus Nerve is One Route of Transneural Invasion for Intranasally Inoculated Influenza A Virus in Mice

Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem. 26 , 34 Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. To evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b) and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. An asymmetric distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side 2 days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The results of this study, thus, demonstrate that neurotropic influenza virus travels to the CNS mainly via the vagus nerve.

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.

Primary target cells of virulent strains of type A influenza virus in chicken embryos

Virulent or avirulent strains of type A influenza virus were inoculated into the allantoic cavities of chicken embryos. The antigens of virulent strains appeared initially in the surface epithelium of the allantoic membrane, then in vascular endothelial cells of the chorioallantoic membrane and visceral organs of the embryos, and then spread to parenchymal cells of many organs. In contrast, the antigens of the avirulent strain were confined to the allantoic membrane. These observations indicate that the primary target of virulent influenza viruses in chicken embryos is vascular endothelial cells, and that the embryos died after systemic viral infection.

Vaccination against H9N2 avian influenza virus reduces bronchus‐associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection

H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin‐inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen‐specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus‐associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin‐inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.