Masaji Mase

Avian Nephritis Virus (ANV) as a New Member of the Family Astroviridae and Construction of Infectious ANV cDNA

ABSTRACT The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family Astroviridae.

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

ABSTRACT The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Complete, Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens

ABSTRACT Mareks disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/β-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.

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.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

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.

Subtyping of Avian Influenza Viruses H1 to H15 on the Basis of Hemagglutinin Genes by PCR Assay and Molecular Determination of Pathogenic Potential

ABSTRACT Serious concern about the worldwide transmission of the Asian H5N1 highly pathogenic (HP) avian influenza (AI) virus by migratory birds surrounds the importance of the AI global surveillance in wild aquatic birds and underscores the requirement for a reliable subtyping method of AI viruses. PCR is advantageous due to its simplicity, lower cross-reactivity, and unlimited reagent supply. Currently, the only available hemagglutinin (HA) subtyping primer set that can subtype H1 through H15 is not fully evaluated and, since it only targets HA1, is unavailable for molecular pathotyping of AI viruses. Our preliminary experiments found that these primer sets were cross-reactive and missed some recent AI viruses. In this study, we developed new primer sets against HA cleavage sites for subtyping H1 to H15 genes and for molecular pathotyping. Our primer sets were subtype specific and detected 99% of previously identified HA genes (115/116, 1949 to March 2006), and the correct amplifications of HA genes were confirmed by sequence analyses of all 115 PCR products. The primer sets successfully subtyped most of the recent AI viruses isolated in Japan (96% [101/105], October 2006 to March 2007). Taken together, our primer sets could efficiently detect HA genes (98% [216/221]) of both previously and recently identified HA genes or of both American (29/29) and Eurasian (187/192) lineages. All 38 H5 and 13 H7 viruses were molecularly pathotyped by sequencing analyses of the HA cleavage site. In contrast, despite efficient detection of previously identified strains (98% [114/116]), the published primer sets exhibited lower specificity and lower detection efficiency against recent AI viruses (80% [84 of 105]). These results indicate that our primers are useful not only for HA subtyping but also for molecular pathotyping of both previous and recent AI viruses. These advancements will enable general diagnostic laboratories to subtype AI viruses for the surveillance in wild aquatic birds.

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.

Identification of a genetic determinant of pathogenicity in chicken anaemia virus

The molecular basis of pathogenicity of the chicken anaemia virus (CAV) needs to be clarified in order to develop a safe, live virus vaccine. In this study, several high- and low-pathogenic infectious DNA clones were obtained from field virus samples after 12 or 38 passages in MDCC-MSB1 cells. The high-pathogenic clones induced a low haematocrit, low weight gain and high mortality. Nucleotide sequence analyses identified one amino acid, at residue 394 of the VP1 capsid protein, as a major determinant of pathogenicity. To determine the role of this amino acid in pathogenicity, chimeric infectious DNA clones and point-mutated clones were used for chicken pathogenicity tests. These analyses clearly demonstrated that residue 394 of VP1 was crucial for the pathogenicity of CAV; all of the cloned viruses with glutamine at this position were highly pathogenic, whereas those with histidine had low pathogenicity. Low-pathogenic CAV, based on an infectious DNA clone, is a candidate for a genetically homogeneous and stable CAV live vaccine.