Susceptibility of BALB/c-nu/nu mice and BALB/c mice to equine herpesvirus 9 infection.

This study aimed to clarify the timing and infectivity of equine herpesvirus 9 (EHV-9) infection in BALB/c-nu/nu mice and their immunocompetent counterpart (BALB/c). Following intranasal inoculation with 10(5) PFU of EHV-9, specimens from 8 mice per group were collected at different times postinoculation (PI) and assessed using histopathology, immunohistochemistry for viral antigen, and quantitative real-time polymerase chain reaction for ORF30 gene expression. In BALB/c-nu/nu mice, EHV-9 antigen was abundant in olfactory epithelia of all inoculated animals, and in the olfactory bulb of 1 animal. In contrast, only 1 BALB/c mouse per time point had rhinitis, with mild to moderate immunopositivity starting from 12 to 48 h PI, followed by a gradual virus clearance at 72 h PI. Statistically, significant differences were noted in the immunohistochemistry reactions between the 2 mouse strains, indicating that BALB/c-nu/nu is more susceptible to infection. Relative expression levels of ORF30 gene in olfactory epithelia were significantly different between the 2 groups, with the exception of 12 h PI, when BALB/c-nu/nu animals showed dramatic increases in ORF30 gene expression level until 48 h PI, followed by a decline in expression level until the end of experiment. In contrast, the expression level in brains showed no differences between mouse strain except at 96 h PI. In both strains, the highest messenger RNA expression was detected at 48 h PI, followed by a decline in BALB/c mice, proving a rapid clearance of virus in BALB/c and a gradual slowing down of the increased expression levels in BALB/c-nu/nu.

Genomic sequence of an infectious bursal disease virus isolate from Zambia: classical attenuated segment B reassortment in nature with existing very virulent segment A.

We determined the complete nucleotide sequence of an infectious bursal disease (IBD) virus (IBDV) isolate (designated KZC-104) from a confirmed IBD outbreak in Lusaka in 2004. The genome consisted of 3,074 and 2,651 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segment A of KZC-104 was derived from a very virulent (VV) strain, whereas its segment B was derived from a classical attenuated strain. On BLAST search, the full-length segment A and B sequences showed 98 % nucleotide sequence identity to the VV strain D6948 and 99.8 % nucleotide sequence identity to the classical attenuated strain D78. This is a unique IBDV reassortant strain that has emerged in nature, involving segment B of a cell-culture-adapted attenuated vaccine.

An ocular infection model using suckling hamsters inoculated with equine herpesvirus 9 (EHV-9): kinetics of the virus and time-course pathogenesis of EHV-9-induced encephalitis via the eyes.

By using a new member of the neurotropic equine herpesviruses, EHV-9, which induced encephalitis in various species via various routes, an ocular infection model was developed in suckling hamsters. The suckling hamsters were inoculated with EHV-9 via the conjunctival route and were sacrificed after 6, 12, 24, 36, 48, 72, 96, 120, and 144 hours (h) post inoculation (PI). Three horizontal sections of the brains, including the eyes and cranial cavity, were examined histologically to assess the viral kinetics and time-course neuropathological alterations using a panoramic view. At 6 to 24 h PI, there were various degrees of necrosis in the conjunctival epithelial cells, as well as frequent mononuclear cell infiltrations in the lamina propria and the tarsus of the eyelid, and frequent myositis of the eyelid muscles. At 96 h PI, encephalitis was observed in the brainstem at the level of the pons and cerebellum. EHV-9 antigen immunoreactivity was detected in the macrophages circulating in the eyelid and around the fine nerve endings supplying the eyelid, the nerves of the extraocular muscles, and the lacrimal glands from 6 h to 144 h PI. At 96 h PI, the viral antigen immunoreactivity was detected in the brainstem at the level of the pons and cerebellum. These results suggest that EHV-9 invaded the brain via the trigeminal nerve in addition to the abducent, oculomotor, and facial nerves. This conjunctival EHV-9 suckling hamster model may be useful in assessing the neuronal spread of neuropathogenic viruses via the eyes to the brain.

Study on the infectivity of equine herpesvirus 9 (EHV-9) by different routes of inoculation in hamsters.

The infectivity and pathology of equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus isolated from gazelles, was studied in hamsters experimentally infected via nasal, ocular, oral, intravenous (IV), or peritoneal routes. Clinically, all animals inoculated by the nasal route and ~25% inoculated by the oral and peritoneal routes showed neurological signs on days 3, 6, and 9 postinoculation (PI), respectively. Neurological signs were not observed in animals administered EHV-9 by the IV and ocular routes. With the exception of animals administered EHV-9 by the IV route, all infected animals had lymphocytic meningoencephalitis. Although there were a number of differences in the severity and distribution of the lesions depending on the route of inoculation, the basic features of lymphocytic meningoencephalitis caused by EHV-9 were common. Lesions consisted of neuronal necrosis, perivascular aggregates of lymphocytes, plasma cells, and neutrophils, gliosis, intranuclear inclusion bodies, and diffuse lymphocytic infiltrates in the meninges. Viral antigen was detected in degenerated neurons in infected animals inoculated by the nasal, ocular, oral, and peritoneal routes. The distribution of EHV-9 antigen was somewhat dependent on inoculation route. There were no microscopic abnormalities or viral antigen in animals treated by the IV route. This study provides new data about experimental EHV-9 infection in hamsters through routes other than the IV route. These results suggest that in the animals infected by the oral, ocular, and peritoneal routes, EHV-9 might travel to the brain through nerves, other than by the olfactory route, after initial propagation at the site of viral entry.

Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Comparative Neuropathogenesis of Equine Herpesvirus 9 and its Mutant Clone (SP21) Inoculated Intranasally in a Hamster Model.

The neuropathogenesis of equine herpesvirus 9 (EHV-9), a neurotropic herpesvirus, and its mutant clone (SP21) was studied experimentally in a hamster model. EHV-9-infected hamsters showed clinical signs of infection at 3 days post infection (dpi), while infection with SP21 resulted in clinical signs at 4 dpi. Clinical signs were more severe in the EHV-9-infected group than in the SP21-infected group. There was a significant difference in the time of anterograde transmission of EHV-9 and SP21 inside the brain. Viraemia was detected in the EHV-9-infected group at 4-5 dpi, while no viraemia was detected in the SP21-infected group. The serum concentration of tumour necrosis factor-α was significantly higher in EHV-9-infected animals than in those infected by SP21 group at 4-5 dpi, but there was no difference in the serum concentration of interferon-γ. The spatiotemporal profiles of viral replication and virus-associated histopathology were remarkably similar, were high in the olfactory bulb and cerebral hemispheres, and decreased progressively towards the medulla oblongata. The mean group scores of the histopathological changes for the entire brain were significantly higher in the EHV-9 group than in the SP21 group at all time points, starting from 3 dpi. These results suggest that the gene products of the open reading frame (ORF)19 and ORF14 play essential roles in the neuropathogenesis of EHV-9, as the two point-mutations detected in SP21 significantly altered the neuropathogenesis of the virus.

Molecular phylogeny of equine herpesvirus 1 isolates from onager, zebra and Thomson's gazelle.

Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy's zebra (Equus grevyi) in 1984 and a Thomson's gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy's zebra and gazelle isolates formed a genetic group that was different from several horse EHV-1 isolates. Within this group, the onager and gazelle isolates were closely related, while the Grevy's zebra isolate was distantly related to these two isolates. The epizootiological origin of the viruses is discussed.

Involvement of multiple Chlamydia suis genotypes in porcine conjunctivitis.

Chlamydia suis has been detected in numerous disease conditions of pigs, particularly in eye infections. This study examined recurring conjunctivitis cases in five commercial pig farms in Japan. 40.5% of the cases were identified as Chlamydia positive using impression cytology of ocular smears and a genus-specific direct fluorescent antibody. C. suis was detected in 59.5% of the samples using PCR tests targeting 16S-23S rRNA intergenic spacer region (ISR) and ompA gene. Genetic analysis of PCR amplicons revealed nine sequence variants of 16S-23S rRNA ISR and 20 sequence variants within ompA gene. Among C. suis-positive conjunctivitis cases, 36.4% showed concurrent infection with 2-4 varied ompA sequence types and 9.1% showed multiple 16S-23S rRNA ISR sequence types of C. suis. Multiple genotypes were found circulating in four of five farms. All 20 detected strains and 25 previously reported C. suis strains were grouped into four clusters. Japanese C. suis strains were closely related to American and European strains indicating wide distribution of these genetically variant strains. This study is the first to show multiple and genetically diverse C. suis strain associations in pig conjunctivitis.

Longer longitudinal atrial dimension in patients with idiopathic paroxysmal atrial fibrillation: A possible cause of atrial fibrillation.

BACKGROUND: The ostium of the superior pulmonary veins or superior vena cava has been reported to be an important source of the ectopic beats that initiate paroxysmal atrial fibrillation (PAF). The structural details of the atria in patients with idiopathic PAF, however, remain unknown. METHODS: We studied 113 patients (92 men and 21 women) with idiopathic PAF and 128 normal control subjects (100 men and 28 women). None of the subjects in either group were found to have any evidence of structural cardiac disease. The echocardiographic measurements were performed in the apical 4-chamber view during end-systole of sinus rhythm. RESULTS: The longitudinal dimension of the left and right atria was longer in patients with PAF who were not administered any drugs (non-drug-taking patients) than in the control subjects (P <.001 and P <.01, respectively). However, there were no significant differences in the transverse dimension of either atrium between such patients and control subjects. The longitudinal and transverse dimensions and volume determinations of atria were greater in the patients with idiopathic PAF who were administered class 1 antiarrhythmic drugs than in non-drug-taking patients (P <.05 to.001). In non-drug-taking patients, prolongation of the atrial longitudinal dimension did not depend on either age, the total frequency of PAF, or the interval from the first episode of PAF. The longitudinal dimension of the left and right atria was longer even in the patients with a short history of PAF (<1 month) as compared with control subjects (P <.001 and.05, respectively). CONCLUSIONS: These observations suggest that there is prolongation of the longitudinal dimension in patients with idiopathic PAF independent of PAF frequency and age (and that PAF is probably a consequence of the prolongation).

Potent dibasic GPIIb/IIIa antagonists with reduced prolongation of bleeding time: synthesis and pharmacological evaluation of 2-oxopiperazine derivatives.

A series of 2-oxopiperazine derivatives, possessing basic moieties at the 3- and the 4-positions, were synthesized and evaluated for their abilities to inhibit platelet aggregation and for their effects on bleeding time. Among the compounds, 2-[(3S)-4-[2-[(4-guanidinobenzoyl)amino]acetyl]-3-[3-[(4-guanidinobenzoyl)amino]propyl]-2-oxopiperazinyl]acetic acid (12c) showed a potent inhibitory effect on platelet aggregation and good dissociation between the efficacy and the bleeding side effect. Intravenous infusion of compound 12c at 1.6 microg/mL/min completely prevented arterial thrombus formation induced by endothelial injury in guinea pigs. The dose of 12c that prolonged the bleeding time to three times the control value was 5.8 microg/mL/min. These results suggest that compound 12c might be useful in the clinical treatment of thrombotic diseases, and we selected 12c (TAK-024) as a candidate for the clinical trials.

Novel non-peptide fibrinogen receptor antagonists. 1. Synthesis and glycoprotein IIb-IIIa antagonistic activities of 1,3,4-trisubstituted 2-oxopiperazine derivatives incorporating side-chain functions of the RGDF peptide.

Based on the lead tetrapeptide RGDF, two possible non-peptide glycoprotein (GP) IIb-IIIa antagonists possessing an (S)-2-oxopiperazine-3-acetic acid moiety as a scaffold incorporating the indispensable Asp fragment were prepared, and (S)-4-[[trans-[4-(guanidinomethyl)-cyclohexyl]carbonyl]glycyl]-2- oxopiperazine-1,3-diacetic acid, 1a, was identified as a potential lead. A series of 3-substituted 2-oxopiperazine-1-acetic acids bearing the Arg-Gly equivalent at the 4-position were prepared and evaluated for their ability to prevent platelet aggregation and for their binding affinity for the GP IIb-IIIa receptor purified from human HEL cells. (S)-4-[(4-Amidinobenzoyl)glycyl]-3-[(methoxycarbonyl)methyl]- 2-oxopiperazine-1-acetic acid, 9 (TAK-029), inhibited in vitro human platelet aggregation with an IC50 value of 0.03 microM and GP IIb-IIIa-fibrinogen binding with an IC50 value of 0.49 nM. The [4-(2-aminoethyl)benzoyl]glycyl derivative 26 showed activity comparable to that of 9 (IC50 = 0.093 microM, guinea pig platelet aggregation assay). Compound 9 dose-dependently inhibited ex vivo platelet aggregation in guinea pigs (0.03 and 0.1 mg/kg, i.v.), and long-lasting inhibition of platelet aggregation was observed upon oral administration of 9 (3 mg/kg) to guinea pigs. On the other hand, the activity of 26 disappeared within 1 h after a dose of 1 mg/kg (i.v.). Compound 9 may therefore be useful in the clinical treatment of arterial thrombotic diseases.

Molecular cloning of an immunogenic and acid-induced isocitrate dehydrogenase gene from Coxiella burnetii.

The Coxiella burnetii icd gene encoding an immunogenic dimeric NADP(+)-dependent isocitrate dehydrogenase (IDH) was cloned by screening a C. burnetii genomic library with a human positive serum and sequenced. The predicted gene product consists of 427 amino acids (M(r) = 46,600) and showed high identity to the IDHs of Escherichia coli (74%), Salmonella enterica (73%) and IDH-I of Vibrio sp. (71%). The cloned gene complemented an icd-defective E. coli mutant producing a recombinant IDH that had the same biochemical properties as the enzyme from purified C. burnetii. Unlike the homologs from other bacteria, the cloned enzyme was expressed to the highest level in low pH conditions. This distinct property of the cloned IDH suggests that C. burnetii icd gene may have a role in the adaptation of the organism to the harsh acidic environment of the eucaryotic phagolysosomes.

Diversity of feline Chlamydia psittaci revealed by random amplification of polymorphic DNA.

DNA samples from C. psittaci including 6 strains of feline origin, 10 strains of avian origin, 1 strain of ovine origin and 1 strain of guinea pig origin were amplified each with three 10-nucleotide (nt) primers and four > 18-nt primers. Amplified products were separated by polyacrylamide gel electrophoresis. Eight patterns were recognized by random amplification of polymorphic DNA (RAPD) fingerprinting of C. psittaci: 2 patterns of feline origin, 5 patterns of avian origin and 1 pattern of guinea pig origin. DNA of feline or guinea pig origin was clearly distinguished from the other strains of C. psittaci by RAPD analysis, as shown by the absence of any common fragments in electrophoresis. The RAPD analysis indicated at least 2 types of feline C. psittaci. The RAPD typing is suggested as a convenient tool for molecular epidemiology of chlamydial infection.

Abortion in Japanese cows caused by Chlamydia psittaci.

An outbreak of abortion in cows occurring in Niigata Prefecture was shown to be caused by Chlamydia psittaci. Elementary bodies characteristic of Chlamydia were found in the liver of aborted fetuses and C. psittaci antigen was demonstrated by indirect immunofluorescence. Chlamydia was isolated from the liver of aborted fetuses by the yolk sac inoculation of developing chick embryos and by the intraperitoneal inoculation of guinea pigs. Abortion occurred mostly in middle or late pregnancy. Aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Focal necrosis was shown in the liver, spleen and lymph nodes. Serological findings and isolation of Chlamydia from fecal specimens indicated a wide dissemination of C. psittaci among cows in the area.

Infectious bursal disease live vaccines: changes in the virus population during serial passage in chickens and chicken embryo fibroblast cells.

Two attenuated infectious bursal disease virus strains used as commercial live vaccine were passaged five successive times in specific-pathogen-free chickens and chicken embryo fibroblast (CEF) cells. Both attenuated strains increased in virulence during the passage in susceptible chickens as evidenced by the decrease in bursa/body weight ratios. A direct nucleotide sequence analysis of the VP2 hypervariable domain amplified by the reverse transcription-polymerase chain reaction revealed that the nucleotide at position 890 (T) in both strains was A after the passage in chicken. In addition, the nucleotide at position 890 (A) was T or C after the subsequent passage in CEF cells. Because of the nucleotide differences, the amino acid residue at position 253 (His) in both vaccines was Gln after the passage in chickens, and the amino acid residue Gln was changed back to His during the subsequent passage in CEF cells. The digestion of the amplified fragment with restriction endonucleases Stul and Ncol, which recognize the sequence difference at position 890, showed that the population of the virus that had amino acid Gln at position 253 was gradually increased during the passage in chickens. Conversely, the population of the virus that had amino acid His at position 253 was gradually increased during the subsequent passage in CEF cells.

In vitro attenuation of highly virulent infectious bursal disease virus: some characteristics of attenuated strains.

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

Invasion and spread of equine herpesvirus 9 in the olfactory pathway of pigs after intranasal inoculation.

The neuropathogenesis of equine herpesvirus 9 (EHV-9) in pigs was investigated by intranasal inoculation of the virus together with intramuscular administration of dexamethasone (DM). All infected pigs developed characteristic meningo-encephalitis, accompanied by basophilic intranuclear inclusion bodies in the neuronal cells. One non-DM-treated and two DM-treated pigs had prominent malacic lesions in the rhinencephalon. Associated with the encephalitic lesions, there was invariably an increase in the number of nucleated cells in the cerebrospinal fluid (CSF). EHV-9 antigen was first detected in the nasal and olfactory epithelial cells in the nasal cavity, and in the neuroglial cells in the olfactory bulb. Subsequently it was demonstrated in the amygdaloid and caudate nuclei, and putamen. The virus was not isolated from the CSF. These results suggest that, after intranasal inoculation, EHV-9 replicates in the olfactory epithelial cells, spreading to the central nervous system via the olfactory pathway.

Experimental infection with equine herpesvirus 9 (EHV-9) in cats.

The pathogenicity for cats of EHV-9, a new neurotropic equine herpesvirus, was assessed by intranasal inoculation with 10(6) plaque-forming units. Four cats killed 4, 5, 6 or 10 days after inoculation showed neurological signs consisting of hyper-excitability and aggressiveness, followed by tremors, occasional convulsions, and depression. Histologically, the cats showed severe encephalitis characterized by neuronal degeneration and loss, intranuclear inclusions, perivascular cuffing and gliosis in the cerebrum. A positive immunohistochemical reaction for EHV-9 antigen was seen in degenerating neuronal cells. The lesions extended from the olfactory bulb to the rhinencephalon and hippocampus. All cats had rhinitis, with or without intranuclear inclusion bodies in the nasal mucosa, and interstitial pneumonia. These findings indicate that the cat, like certain other species such as the goat, is susceptible to experimental infection with EHV-9, and may be at risk from natural infection.

Neuropathological study of gazelle herpesvirus 1 (equine herpesvirus 9) infection in Thomson's gazelles (Gazella thomsoni).

Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomson's gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.

The prevalence of bovine herpesvirus-1 in traditional cattle in Southern Province, Zambia.

A study was conducted to determine the prevalence of bovine herpesvirus-1 (BHV-1), which causes infectious bovine rhinotracheitis, in cattle destined for market in Southern Province, Zambia. A total of 116 nasal secretion samples were tested using the direct fluorescent antibody test, while blood samples from the same cattle were examined by a commercial enzyme-linked immunosorbent assay kit. The prevalence of the BHV-1 antigens in cattle was 23.28% (27/116), while the mean prevalence of the BHV-1 antibodies was 48.28% (56/116). This study showed that cattle in transit to markets could easily spread the virus, which was reactivated by the stress of trekking for long distances under unfavourable conditions, to the other cattle with which they came into contact. Thus, these transit cattle posed a serious threat to other bovines. Systems of cattle trading where cattle must be transported a long wayto market should be reviewed by the authorities to minimise the conditions that may exacerbate the spread of infection.