Capsid protein gene variation among feline calicivirus isolates.

We amplified the capsid protein gene fragments of 30 Japanese isolates of feline calicivirus (FCV), including the C, D, and E regions, by reverse transcription-polymerase chain reaction (RT-PCR), followed by direct sequencing. Alignment of the predicted amino acid sequences, together with other published sequences from the isolates obtained in other countries, demonstrated a marked heterogeneity among the isolates, confirming the current definition of hypervariable regions within the capsid protein: these regions give rise to the antigenic variations seen in FCV isolates. Phylogenetic analysis of the nucleotide sequences could not identify significant geographically or temporally separated clusters of FCV isolates, supporting the theory of a single genotype.

Identification of the porcine cytomegalovirus major capsid protein gene.

A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified. Sequence analysis indicated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues. The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts. Phylogenetic analysis using herpesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse cytomegaloviruses. The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection. Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection. The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving virus-isolation are not useful.

Immunological and PCR analyses for Borna disease virus in psychiatric patients and blood donors in Japan.

The involvement of Borna disease virus (BDV) in psychiatric diseases in humans remains controversial. T-cell memory response and seroprevalence of BDV in patients with psychiatric disorders and blood donors in Japan were evaluated collectively by Western blot (WB) analysis with inhibition test, electrochemiluminescence immunoassay, immunofluorescence assay, and T-cell proliferative response as well as detection of BDV p24 RNA in peripheral blood mononuclear cells (PBMCs). Positive proliferative responses to both BDV p40 and p24 proteins were detected in 9% of patients with mood disorders (4 of 45), 4% of schizophrenic patients (2 of 45), and 2% of blood donors (1 of 45). By WB analysis, the antibody to BDV p40 was detected only in 2% of patients with mood disorders (1 of 45). The BDV p24 antibody was detected in 2% of patients with mood disorders (1 of 45) and 9% of schizophrenic patients. (4 of 45) No plasma reacted with both BDV proteins. The finding of a lower seroprevalence than previously reported suggests the presence of false-positive cases in the previous report. BDV RNA was detected only in 2% of patients with mood disorders (1 of 45). In these three serological assays, T-cell responses, and PCR analysis, there was no significant difference in the prevalence among the three groups. However, we found three psychiatric patients who were positive for both BDV antibodies and T-cell proliferative responses and one patient who was positive for BDV RNA in PBMCs. These findings suggest the usefulness of the proliferative T-cell response and that certain individuals are infected with BDV or a BDV-related virus.

Pandemic threat posed by avian influenza A viruses.

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.

Recognition of N-glycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks.

The hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing N-glycolneuramic acid (NeuGc) linked to galactose by the alpha2,3 linkage (NeuGcalpha2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGcalpha2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGcalpha2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses.

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR.

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.

Seroepidemiological survey of feline retrovirus infections in domestic and leopard cats in northern Vietnam in 1997.

The prevalence of infections with three feline retroviruses (feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and feline syncytial virus (FSV)) was examined in northern Vietnam in 1997. We collected a total of 77 blood samples from 69 domestic and 8 leopard cats, and examined the presence of anti-FIV and FSV antibodies and FeLV p27 antigen in the plasma samples by the indirect immunofluorescence and/or two commercial kits. None of the samples was positive for FIV and FeLV. The overall positive rate of FSV was 31% and the positive rates among the domestic and leopard cats were 29 and 50%, respectively. We isolated FSV from peripheral blood mononuclear cells of 6 domestic and one leopard cats.

Properties and functions of feline herpesvirus type 1 glycoproteins.

Feline herpesvirus type 1 (FHV-1) is a causative agent of feline viral rhinotracheitis and belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. Since first isolated in 1958 by Crandell and Maurer, FHV-1 is distributed worldwide and is the most clinically significant agent for respiratory infections in cats. In this review, we describe the recent findings with properties and functions of FHV-1 glycoproteins, especially hemagglutinins.

A possible mechanism for selection of virulent avian influenza A viruses in 14-day-old embryonated eggs.

The emergence of virulent avian influenza viruses in poultry is unpredictable. To gain insight into the mechanism of this event, we previously examined the possible role of older (14-day-old) embryonated eggs, in which virulent mutants were preferably selected (Horimoto and Kawaoka, Virology 206: 755-759, 1995). However, it is unknown why virulent mutants replicate predominantly in older eggs. In the present study, we compared protease activities responsible for cleavage activation of the hemagglutinin (HA) in allantoic fluids in 10-day and 14-day-old eggs. In vitro assays showed that the protease activities were stronger in the 14-day-old than 10-day-old eggs. The allantoic fluids with strong protease activity degraded HA. These results indicate that replication of avirulent viruses is hampered in older eggs, while that of virulent viruses whose HAs are activated by other intracellular proteases was not, possibly leading to a replicative advantage for virulent mutants in the older eggs.

A reverse-type sandwich enzyme-linked immunosorbent assay for detecting antibodies to Borna disease virus.

To investigate whether there is an epidemiological correlation between Borna disease virus (BDV) infection and human neuropsychiatric diseases, we established a reverse-type sandwich enzyme-linked immunosorbent assay (RS-ELISA) for detecting specific antibodies to BDV. In this assay, microplate wells were coated dispersely with BDV p40 antigen, followed by the addition of test samples at a low dilution and then the biotinylated p40. A preformed complex of streptavidin and horseradish peroxidase-conjugated biotin and an enzyme substrate were used to measure the captured biotinylated p40. Theoretically, RS-ELISA should specifically detect anti-BDV antibodies without nonspecific signals; such signals possibly occur in conventional serological assays. Additionally, the RS-ELISA could be applied under the same protocols to test samples from a variety of animals. By using anti-BDV rat and rabbit sera, the assay was standardized so that it had high specificity and sensitivity. When we used the RS-ELISA to determine the presence of anti-BDV antibodies in plasma from 70 patients with chronic schizophrenia as well as 40 healthy individuals in the Tokyo area of Japan, no plasma sample was found to possess specific antibodies to BDV p40, indicating no association between BDV infection and the disease in our testing population. A negative reaction was also shown for the sera that had previously been judged to be seropositive for BDV by an immunofluorescence or immunoblot test. These findings suggested that false-positive cases of infection due to nonspecific reactions may be included in previous seroepidemiological information with regard to BDV.

Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus.

We mutated the virulent avian influenza virus A/turkey/Ontario/7732/66 (H5N9)[Q-R-R-R-K-K-R\G at the hemagglutinin (HA) cleavage site] to create a mutant, R(MO-0), with additional basic residues at this site (Q-R-R-R-R-R-K-K-R\G) by reverse genetics. When tested in chicken embryo fibroblast culture, this mutant showed reduced HA cleavability compared to that of the wild-type virus, but its plaque size was not appreciably altered. Virulence of the R(MO-0) virus in chickens was lower than that of the wild-type virus. These findings indicate that addition of excessive basic residues to an optimal recognition sequence for HA cleavage enzymes at the cleavage site is deleterious for HA cleavability. Previously, we showed that a mutant containing the suboptimal HA cleavage site sequence for cleavage enzyme recognition also had reduced HA cleavability and virulence compared to the wild-type virus. We conclude that the data presented here further substantiate our belief that the level of HA cleavability correlates with the degree of virulence when all other genetic characteristics are considered equal, irrespective of the mechanisms by which HA cleavability is reduced.

Characterization of several pseudorabies viral strains by virus-neutralization test using monoclonal antibodies.

In the present study, five mouse monoclonal antibodies (MAbs) to the pseudorabies virus (PRV) Yamagata-81 strain were produced. The MAbs were used in cross-neutralization tests and cross-indirect enzyme-linked immunosorbent assay (ELISA) against three PRV viral strains isolated in Argentina and another four obtained from the United States, Japan, France, and Sweden. Four of five MAbs needed the presence of complement to produce or enhance neutralization activity. No differences were observed by ELISA. The MAbs showed different neutralizing activity against PRV strains, suggesting phenotypic heterogeneity among them.

Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR.

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

[Laboratory virological diagnosis of imported dengue cases].

Suspected dengue cases in Japan during the period of 1985-1995, 173 cases with unknown febrile illness entering or returning from mostly southeast Asia, were tested by serology and RT-PCR gene amplification. Seventy seven cases were confirmed by a significant rise of dengue 2 (Den 2) HI antibody in paired sera or by detection of HI antibody titer higher than 1:320 in single serum. The other 3 cases with antibody levels not higher than 1:80 in paired sera and 12 cases with an antibody 1:160 in single sera were positively suspected of dengue infection but were not confirmed. Countries of origin of confirmed cases were Thailand (39 cases), Philippinse (15), India (13), and Indonesia (9). Sera of dengue cases showed high degrees of cross reactivity of Japanese encephalitis virus (JEV) in HI test but not in IgM capture ELISA. Sera of confirmed JEV-infected cases, however, showed practically no cross reactivity to Den 1 4 in HI test, suggesting unilateral cross reactivity of HI antibody. RT-PCR detected the Den 1 genome in sera of 3 cases obtained within 3 days after onset and the Den 2 genome in serum of case 4 days after onset. Although the number is limited, 92 (53%) out of 173 cases of febrile illness of unknown etiology entering Japan from tropical countries were either confirmed or positively suspected to be dengue fever. Considering possibilities of under reporting, importations of tropical viral infections should be bigger in number and will necessitate our intensified alertness.

Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses.

Lesions of chickens inoculated with two highly pathogenic avian influenza virus strains, A/turkey/England/50-92/91 (H5N1) and A/chicken/Victoria/1/85 (H7N7) were examined histologically and immunohistochemically. Birds of both treatment groups died within 5 days post-inoculation. The most significant lesions induced by these two viruses consisted of swelling of the microvascular endothelium, systemic congestion, multifocal haemorrhages, perivascular mononuclear cell infiltration, and thrombosis associated with viral antigen in the vascular endothelium and/or perivascular parenchymatous cells. Viral antigen in the cardiac myocytes was consistently detected in all birds. In addition, severe pulmonary congestion and oedema was found in A/turkey/England/50-92/91 virus-inoculated birds that died within 1 day post-inoculation. The other chickens of both groups showed necrotic and inflammatory changes associated with viral antigen in various organs and tissues. These findings suggested that cardiovascular system involvement played an important role in the pathogenesis of these virus infections.

Neuropathological studies of chickens infected with highly pathogenic avian influenza viruses.

Central nervous system lesions of chickens inoculated with three highly pathogenic avian influenza virus strains, A/chicken/Victoria/1/85 (H7N7), A/turkey/England/50-92/91 (H5N1), and A/tern/South Africa/61 (H5N3), were examined histologically and immunohistochemically. The chickens either died within 7 days of inoculation or were killed 2 weeks after inoculation. No significant differences were observed in the lesions induced by these three viruses. The lesions were divided into two types, disseminated foci of microgliosis and necrosis, and ventriculitis. The former lesions were associated with infection of the vascular endothelium and dissemination of the virus to the peripheral parenchymal cells of the chickens that died within 3 days of inoculation. The ventriculitis lesions, however, were observed mainly in the chickens that died between 4 and 7 days after inoculation. These findings suggest that viral infection of the vascular endothelium and subsequent involvement of ependymal cells play important roles in the pathogenesis of the central nervous system lesions.

Cleavability of hemagglutinin from an extremely virulent strain of avian influenza virus containing a unique cleavage site sequence.

An avian influenza virus, A/turkey/England/50-92/91 (H5N1), showed extremely high virulence in chickens, although its hemagglutinin (HA) cleavage site sequence (R-K-R-K-T-R), having a nonbasic (Thr) residue at the second position (P-2) from the carboxyl terminus of HA1, does not conform to the previously established consensus sequence motif, X-X-R/K-X-R/K-R (X = nonbasic residue), for highly virulent phenotype of the H5 virus. When we evaluated the HA cleavability of this strain in chicken embryo fibroblast culture, we observed that, unlike other HAs with a Thr residue at P-2, this HA was efficiently cleaved. These findings suggest that a nonbasic residue at the P-2 does not affect its recognition and catalyzation by cleavage enzymes that are otherwise influenced by steric structure around the cleavage site.