Kunitoshi Imai

Phylogenetic Analysis of Newcastle Disease Virus Genotypes Isolated in Japan

ABSTRACT We genetically analyzed field isolates of the Newcastle disease (ND) virus isolated in Japan from 1930 to 2001. The coding region of the fusion protein was amplified by reverse transcriptase PCR and directly sequenced. Phylogenetic analysis revealed the presence of viruses belonging to six of the eight known genotypes. It can be concluded from this study that ND outbreaks in Japan have been of multiple etiologies.

Efficacy of three live vaccines against highly virulent infectious bursal disease virus in chickens with or without maternal antibodies.

Since 1990, highly virulent infectious bursal disease virus (IBDV), which induces high mortality, has been infecting even vaccinated flocks in Japan. We report the efficacy of three live vaccines that are available in Japan. Two mildly attenuated strains (A and B) and one intermediate strain (C) were each tested both in specific-pathogen-free (SPF) chickens and in commercial chickens that have maternal antibodies against IBDV. Chickens were vaccinated at 20 days old and challenged with highly virulent IBDV 10 days post-vaccination. Protection was determined 7 days after challenge by measuring bursa/body weight ratios, histopathological lesions, and antibody responses to IBDV. All three lie vaccines conferred protection to SPF chickens. However, only vaccine C protected 100% of vaccinated commercial chickens against highly virulent IBDV; Vaccines A and B respectively protected three-fourths and none of vaccinated commercial chickens from severe bursal lesions. Vaccines A, B, and C and highly virulent IBDV induced bursal lesions in 3%, 0%, 23%, and 61% of inoculated commercial chickens, respectively. These results suggest that serological determination of the optimum vaccination time for each flock is required to effectively control highly virulent IBDV in the field. The optimum vaccination timing could be approximated by titrating the maternal IBDV antibodies of 1-day-old chicks by an enzyme-linked immunosorbent assay or by an agar gel precipitin test.

Pathology of Fatal Highly Pathogenic H5N1 Avian Influenza Virus Infection in Large-billed Crows (Corvus macrorhynchos) during the 2004 Outbreak in Japan

Highly pathogenic H5N1 avian influenza viruses were isolated in 9 large-billed crows that died in Kyoto and Osaka prefectures in Japan from March to April in 2004. We studied 3 of the 9 crows using standard histologic methods, immunohistochemistry, and virus isolation. The most prominent lesions were gross patchy areas of reddish discoloration in the pancreas. The consistent histologic lesions included severe multifocal necrotizing pancreatitis, focal degeneration and necrosis of neuron and glial cells in the central nervous system, and focal degeneration of cardiac myocytes. All of these tissues contained immunohistochemically positive influenza viral antigens. The virus was isolated from the brain, lung, heart, liver, spleen, and kidney of the crows examined. Thus we concluded that highly pathogenic avian influenza virus was associated with clinical disease, severe pathologic changes, and death in the 3 crows.

Mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus.

Hydroxytyrosol (HT), a small-molecule phenolic compound, inactivated influenza A viruses including H1N1, H3N2, H5N1, and H9N2 subtypes. HT also inactivated Newcastle disease virus but not bovine rotavirus, and fowl adenovirus, suggesting that the mechanism of the antiviral effect of HT might require the presence of a viral envelope. Pretreatment of MDCK cells with HT did not affect the propagation of H9N2 virus subsequently inoculated onto the cells, implying that HT targets the virus but not the host cell. H9N2 virus inactivated with HT retained unaltered hemagglutinating activity and bound to MDCK cells in a manner similar to untreated virus. Neuraminidase activity in the HT-treated virus also remained unchanged. However, in the cells inoculated with HT-inactivated H9N2 virus, neither viral mRNA nor viral protein was detected. Electron microscopic analysis revealed morphological abnormalities in the HT-treated H9N2 virus. Most structures found in the HT-treated virus were atypical of influenza virions, and localization of hemagglutinin was not necessarily confined on the virion surface. These observations suggest that the structure of H9N2 virus could be disrupted by HT.

Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains.

In order to study replication efficiency of infectious bursal disease virus (IBDV) in lymphoid tissues, both the virus titers and the virus antigen titers in four lymphoid tissues were compared among chickens inoculated with Ehime/91 (about 50% mortality), J1 (no mortality), or K (attenuated) IBDV strains during 1-7 days postinoculation (PI). IBDV antigens in tissue homogenates were detected by an enzyme-linked immunosorbent assay. In the bursa of Fabricius, higher virus titers were maintained for 1-3 days PI in chickens inoculated with Ehime/91 or J1 strains, whereas the virus titers increased gradually and reached to the peak on 3 days PI in chickens inoculated with the K strain. There were no clear differences in both the virus and the virus antigen titers in bursae and thymus between chickens inoculated with Ehime/91 and J1 stains. However, the virus and/or the virus antigen titers in spleen and bone marrow of chickens inoculated with Ehime/91 strain were higher than those of the J1-inoculated chickens. Immunohistochemical analyses indicated that larger numbers of IBDV antigen-positive cells were detected in spleen and bone marrow of the Ehime/91 group than in those of the J1 group. There was almost no detectable virus and virus antigens in thymus, spleen, and bone marrow of the K-inoculated chickens throughout the experiment. These results suggest that efficient replication of IBDV not only in the bursa but also in the spleen and the bone marrow may be required for clinical infectious bursal disease.

Pathology of Specific-Pathogen-Free Chickens Inoculated with H5N1 Avian Influenza Viruses Isolated in Japan in 2004

Abstract Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1–4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials

The effect of cotton textiles containing Cu(2+) held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1 ml) was applied to the textile (3×3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9 ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin-Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs. The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log(10) and 5.0 log(10), respectively, in 30 s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5 min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10 min, the titer declined by >5.0 log(10) in MDCK cells and by >3.5 log(10) in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus-receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu(2+) in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu(2+). Interestingly, CuCl(2) treatment (500 and 5000 μM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48 h of incubation, although the titer of the purified H5N3 virus treated with CuCl(2) declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl(2) treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.

Pathologic and Immunohistochemical Studies of Newcastle Disease (ND) in Broiler Chickens Vaccinated with ND: Severe Nonpurulent Encephalitis and Necrotizing Pancreatitis

Twenty-five 22– to 46–day-old broilers with Newcastle disease (ND) were investigated pathologically and immunohistochemically in order to evaluate the mechanism of ND outbreak in vaccinated broilers. The broilers were vaccinated with ND live vaccine via drinking water. Clinical signs were neurologic and respiratory in nature. Macroscopically, bursal atrophy, white spots on the pancreas, and discoloration and enlargement of kidneys and spleen were observed in the broilers. Histologically, perivascular cuffing, neuronal degeneration and necrosis, and glial proliferation were present in the cerebrum, cerebellum, and medulla oblongata. There was extensive rarefaction and malacia in the parenchyma of severely affected brains. There were extensive degeneration, necrosis, and depletion of acinar cells in the pancreas. There was proliferation of macrophages in the lungs with congestion, tubulointerstitial nephritis, hepatocytic necrosis with thrombi in the sinusoids, and lymphocytic depletion in the cloacal bursa. Immunohistochemically, ND virus antigens were detected in the lesions. ND virus isolated from the present cases did not cause encephalitis or pancreatitis in specific-pathogen-free chickens, but it induced mortality with hepatocytic sinusoidal thrombi, splenic necrosis, lymphoid necrosis and depletion, and conjunctival hemorrhage. Severe nonpurulent encephalitis with extensive rarefaction and malacia, and necrotizing pancreatitis in the present case may suggest a close possibly causal relation with vaccination.

Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread.

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

Presence of Avian Pneumovirus Subtypes A and B in Japan

Abstract Four avian pneumovirus (APV) isolates from chickens clinically diagnosed with swollen head syndrome were genetically characterized as to the subtypes of the virus in Japan. The results of reverse transcriptase–polymerase chain reactions based on subtype-specific primers and direct sequence analysis of G genes indicated subtypes A and B but not C or D of APV were present in Japan. Several routes or sources are conceivable for APV to invade into Japan.