Physicochemical properties of akabane virus: A member of the simbu arbovirus group of the family bunyaviridae

The multiplication of Akabane virus was not inhibited in the presence of 5-iodo-2′-deoxyuridine, indicating the presence of RNA. The virus was considered to have an envelope, as it was sensitive to ether and chloroform. It was readily inactivated by deoxycholate and trypsin, but was not precipitated by protamine sulphate. The virus was very labile at pH 3 and also rather heat-labile. Akabane virus was readily filtered through membrane filters of 200 or 100-nm pore size, but not through 50-nm filters. Equilibrium centrifugation in a CsCl density gradient gave a peak of infectivity and hemagglutinin at a density of 1.22 g/ml. The peak fractions thus obtained contained numerous virus particles, roughly spherical, variable in size, 70 to 130 nm in diameter, and mostly having a ragged, closely adherent envelope with projections, when examined, following phosphotungstic acid negative staining, in an electron microscope.     

Properties of a coronavirus isolated from a cow with epizootic diarrhea

A coronavirus (Kakegawa isolate) isolated from a cow with epizootic diarrhea was grown in BEK-1 cells and examined for biophysical and biochemical properties. The Kakegawa isolate was able to replicate in the presence or absence of 5-iodo-2′-deoxy-uridine, indicating that its viral nucleic acid was RNA. It was highly sensitive to ether and chloroform, and moderately sensitive to trypsin and heat. It was, however, readily stabilized by treatment with cation at 50°C for 1 h. Its infectivity was slightly reduced at pH 3.0. The virus passed through a membrane filter of 200 nm pore size, but not through one of 100 nm pore size. The buoyant density of the virus was determined in a sucrose density gradient. The peak of infectivity and hemagglutinin activity was found at a density of 1.182. Neutralization and hemagglutination inhibition tests showed a close serological relationship between the Kakegawa isolate and the American strain of calf diarrhea coronavirus.     

Physicochemical Characterization of a Neonatal Calf Diarrhea Virus

Physicochemical characteristics of two isolates of a neonatal calf diarrhea virus were investigated. Neither isolate was sensitive to ether or chloroform, both were stable at pH 3.0, were relatively heat resistant, but were thermolabile when heated to 50°C for one hour in the presence of 1.0 M MgCl₂. Multiplication of virus was not inhibited by concentrations of 5-iodo-2'-deoxyuridine (IDUR) up to 500 µg/ml, which indicated that the nucleic acid was ribonucleic acid (RNA). Also, multiplication was not inhibited by concentrations of actinomycin D (AD) up to 0.5 µg/ml. Thermal denaturation studies demonstrated that the nucleic acid had a high melting temperature.

Resistance to lipid solvents, stability at an acid pH, relatively high thermostability, type of nucleic acid, plus previous reports from this laboratory on general morphology and cytopathogenicity suggest that the virus may belong to the diplornavirus (reovirus) group. However, thermolability in the presence of 1.0 M MgCl₂ is not consistent with characteristics of this group.

     

Moraxella bovis hemolysin

Moraxella bovis hemolysin was readily filterable through polycarbonate membrane filters, but not through nitrocellulose filters. The hemolysin was filterable through polycarbonate filters with pore diameters of greater than or equal to 0.015 micron (APD). Of the hemolytic activity of cell-free filtrates, 74% could be pelleted by ultracentrifugation at 100,000 X g for 2 1/2 hours. Hemolytic activity could be demonstrated in preparations of outer membrane fragments isolated from log-phase cultures. Hemolysin in M bovis broth cultures reached a maximum concentration in late logarithmic phase (4.5 hours after inoculation) and declined thereafter. Hemolysin was inactivated by heat, trypsin, formalin, and lyophilization.     

The requirement of environmental acidification for Ibaraki virus infection to host cells

The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. IBAV outer capsid proteins, VP5 and VP2, were removed from virion when purified IBAV was exposed to low pH environment. Further experiment showed that the exposure to low pH buffer facilitated IBAV infection when the cellular endosomal pathway was impaired by bafilomycin A1. Results obtained in this study suggest that acidic environment is essential to initiate IBAV infection.     

Infectious dose‐dependent accumulation of live highly pathogenic avian influenza H5N1 virus in chicken skeletal muscle—implications for public health

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross‐boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non‐fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (10², 10³ and 10⁴ EID₅₀) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 10² EID₅₀) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post‐infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses.     

Viral shedding and emission of airborne infectious bursal disease virus from a broiler room

1. The significance of airborne transmission in epidemics of infectious diseases in the livestock production industry remains unclear. The study therefore investigated the shedding route (faeces vs. exhaled air) of a vaccine strain of infectious bursal disease virus (IBDV) by broilers and the emission of airborne virus.

2. The experimental room contained 526 broilers which were orally inoculated at the age of 20?d. The airborne virus was sampled by three different bioaerosol samplers: Andersen six-stage impactor, all-glass impinger (AGI-30) and OMNI-3000.

3. Infected broilers started to shed virus in faeces on d 5 post inoculation (PI), and stopped shedding on d 12 PI. The faecal virus remained detectable for at least two d after drying under broiler room conditions. No virus was detected in the air exhaled by broilers.

4. Airborne virus was collected on d 5, 8 and 12 PI at 20?cm above the floor, and on d 8 and 12 PI in exhausted air. The emission rates of IBDV were 4·0 log₁₀ 50% tissue culture infectious dose (TCID₅₀)/bird/d on d 8 PI, and 4·5 log₁₀ TCID₅₀/bird/d on d 12 PI.

5. We concluded that broilers shed IBDV mainly through their faeces. The presence of indoor airborne virus is associated with the viral presence in faeces. The successful recovery of airborne virus in exhausted air indicates there is a potential risk of virus spreading to the ambient environment via air.     

Characterization of a caprine herpesvirus.

A virus, which was isolated from kids (Capra hircus) affected with a relatively severe generalized infection, was found to contain DNA and to have a buoyant density of 1.2820 g/cm3. The virus was sensitive to the action of lipid solvents and trypsin and was rapidly inactivated at pH 3.0 and at temperatures of 50 and 56 C. The virion, an icosahedron consisting of a nucleoid surrounded by a double membrane, measured approximately 135 nm in diameter. On the basis of its chemical and physical properties, the virus is considered a herpesvirus.     

Humoral immune response of calves to bluetongue virus infection

Humoral immune responses of 7 calves to bluetongue virus (BTV) infection were evaluated by plaque-reduction assay and immunoblotting. Most readily interpretable results were obtained with the immunoblot assay when colostrum-deprived calves were used, and sera were reacted with proteins in partially purified extracts of BTV. Viremia persisted in calves for 35 to 56 days, and BTV coexisted in blood for several weeks with virus-specific neutralizing antibody. Calves developed antibody to virus protein 2, the major determinant of virus neutralization, at 14 to 28 days after inoculation; this time interval also coincided with the appearance of neutralizing antibody in serum. Virus clearance in BTV-infected calves did not coincide with humoral immune responses to protein 2 or other virion proteins.     

Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry, suggesting its potential to become a pandemic influenza virus in humans. The transmission route of the virus among either the same species or different species is not yet clear. Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds. The animals were inoculated with 0.1 mL 10^6.83 TCID₅₀ of H5N1 virus oronasally, intraperitoneally and using eye drops. The viruses were examined by virological and pathological assays. In addition, to detect horizontal transmission, in each group, healthy chicks and mice were mixed with those infected. Viruses were detected in homogenates of the heart, liver, spleen, kidney and blood of the infected mice and chickens. Virus antigen was not detected in the spleen, kidney or gastrointestinal tract, but detected by Plaque Forming Unit (PFU) assay in the brain, liver and lung without degenerative change in these organs (in the group inoculated using eye drops. The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice. All chicken samples tested positive for the virus, regardless of the method of inoculation. Avian influenza A H5N1 viruses are highly pathogenic to chickens, but its virulence in other animals is not yet known. To sum up, the results suggest that the virus replicates not only in different animal species but also through different routes of infection. In addition, the virus was detection not only in the respiratory tract but also in multiple extra‐respiratory tissues. This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.     

A Study of Bovine Virus Diarrhea Mucosal Disease Virus by Plaque Technique

Bovine virus diarrhea-mucosal disease (BVD-MD), NADL, strain formed 3-4 mm plaques on monolayers of bovine embryo kidney (BEK), lung and testicular (BET) cell cultures on post inoculation day four. Plaques were 1.5 mm on the post inoculation day five in lamb testicular cell cultures. Neutral red incorporated in first overlay had inhibitory effect on plaque formation in these cell-virus systems. The study of effects of environmental variables on plaquing efficiency indicated that virus adsorption rate was temperature dependent and approximately 80% virus was adsorbed onto BET monolayers in two hours. Rate of adsorption was slightly superior in BEK monolayers than the ones recorded in BET cell cultures. Virus diluent should contain calcium and magnesium ions for maximum plaquing efficiency. Cultures maintained under lamb serum should be washed for the development of maximum number of plaques. Virus particles could diffuse through agar overlay to initiate infection and form delayed plaques. Size of the plaques was proportional to the concentration of agar in overlay medium. Plaquing efficiency was also dependent upon pH of the overlay and optimum pH for maximum efficiency was 7.3 - 7.7.

NADL strain of BVD-MD virus was sensitive to trypsin but resistant to 5'-Bromodeoxyuridine. Thermostability studies showed that 0.5% virus survived when incubated at 37°C for 48 hours. The virus was sensitive to freezing and thawing.

Comparative titers of virus determined and expressed as PFU and TCID₅₀ were almost similar.

     

THE ISOLATION OF PICORNAVIRUSES FROM CATS WITH RESPIRATORY DISEASE

One hundred and sixty samples including nasal swabs, conjunctival swabs, lung tissue, swabs of tonsil and an oral swab were taken from 59 cats suffering from respiratory disease. Thirty-nine of the specimens (from 23 cats) contained agents that produced a rapid cytopathic effect in cultures of feline kidney cells. Two of the isolates were studied in detail, and they were found to be resistant to lipid solvents, stable at pH 5 but not at pH 3, able to pass through a 50 mμ filter and not stabilized to inactivation at 50°C in the presence of molar concentrations of MgCl₂. Electron microscopy of negatively stained preparations revealed particles about 45 mμ in diameter that lacked an outer envelope. These properties indicate that the agents are feline picornaviruses.
Antibodies to one of the isolates were present in 14 of 56 cat serums. Four kittens inoculated with picornaviruses developed upper respiratory tract disease, but 3 died with signs of enteritis possibly caused by concurrent infection with pan-leucopenia virus. Picornaviruses were isolated from nasal swabs from 3 of these kittens, and from lung and spleen of the other.     

The Purification and Concentration of Hog Cholera Virus with Electron Micrographs: (A Preliminary Note)

Cell-cultured hog cholera virus was partially purified by chromatography on pulverized magnetic ferric oxide. Infectivity yields of 50 to 100% were obtained with a 90 to 95% reduction in extraneous organic nitrogen. Concentration and further purification of infectious virus were achieved by rate-zonal and isopycnic ultra-centrifugations in buffered cesium chloride. Density determinations obtained from the isopycnic experiments indicate a buoyant density of 1.14-1.15 gm/ml for the strain of virus used. Electron microscopy of negatively stained samples of concentrated infective virus from one isopycnic experiment revealed 40- to 40-mµ virus-like particles and a large number of 12- to 15 mµ entities. The 40- to 50-mµ particles were surrounded by a poorly defined, asymmetrically arranged sac-like membrane or appendage. It is suggested that the images of the 40- to 50-mµ particles represent the infective virion of hog cholera virus.     

Hemolytic activity of Akabane virus

Akabane virus was shown to lyse as well as to agglutinate pigeon erythrocytes. The hemolytic activity of the virus was markedly enhanced by repeated freeze-thawing, but its hemagglutinating activity was not affected. Hemolysis (HL) with the virus, like its hemagglutination, was affected by the NaCl concentration as well as by the pH of the diluent. HL was markedly affected by the incubation temperature, but hemagglutination (HA) was not; HL activity was highest at 37°C, somewhat lower at 25°C, very low at 4°C, and did not occur at 0°C. While pigeon erythrocytes were positive for both HL and HA, goose erythrocytes were positive for HA but negative for HL. Erythrocytes from cattle, sheep, rabbits, guinea pigs, mice and day-old chickens were tested for HA as well as for HL activity with negative results. A linear relationship was shown, in a wide range of the virus concentrations, between the percent HL and the virus concentration, as expressed on a logarithmic scale. Based on these findings we developed an assay method for Akabane virus hemolysin. Analysis by CsCl equilibrium density gradient centrifugation indicated the hemolytic as well as the hemagglutinating activity to be structurally associated with the virion. Scanning electron microscopy of pigeon erythrocytes undergoing HL with the virus revealed the appearance of a depressed area with a hole on the cell surface. The hemolytic activity of the virus was specifically inhibited by antisera to the virus and an HL-inhibition test was developed.     

Isolation and preliminary characterization of an orbivirus of the Palyam serogroup from biting midge Culicoides oxystoma in Japan

An orbivirus of the Palyam serogroup was isolated from Culicoides oxystoma collected in a cowshed in Kagoshima, Southern Kyushu Island, Japan. This is the first isolation of an orbivirus of the Palyam serogroup in Japan. The virus was a spherical non-enveloped RNA virus, approximately 60 nm in diameter. The virus was resistant to ethyl ether, sodium deoxycholate and freezing-thawing, but readily inactivated by trypsin. The virus was not stabilized by 1 M MgCl2, was labile at pH 3.0 and was not precipitated by protamine sulfate. Indirect immunofluorescent staining of infected Vero cells indicated the virus to be antigenically related to D'Aguilar and Bunyip Creek viruses of the Palyam serogroup. Neutralization tests showed the virus to have no relationship with D'Aguilar virus, but to have a one-way cross-reaction with Bunyip Creek virus. The virus was tentatively designated as Kagoshima virus. A serological survey indicated dissemination of the virus in cattle populations in Kagoshima Prefecture.     

Chicken infectious anemia in Mexico: virus identification and serology survey.

Three chicken infectious anemia (CIA) virus strains were isolated from 10 different sick broiler and replacement chicken flocks with the MDCC-MSB1 cell line. One-day-old specific-pathogen-free chicks were inoculated later, with the three original samples being positive in tissue culture; one induced signs and lesions, another only lesions typical for CIA. One isolate was selected for further trials and showed resistance to chloroform and heat (75 C for 5 min) and passed through a 45-nm filter membrane but did not pass through the 22-nm filter. These characteristics were similar to the Del Rose reference strain of chicken anemia virus. By electron microscopy, the diameter of particles obtained from the pellet of infected cell cultures was between 22 and 27 nm. Serology survey carried out with 580 serum samples from different poultry farms all over the country with a commercial enzyme-linked immunosorbent assay kit gave proof of widespread seroconversion, indicating that CIA should be considered endemic to Mexico.     

Physicochemical properties of pseudorabies virus hemagglutinin.

Pseudorabies virus hemagglutinin was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on cattle erythrocytes. The adsorbed hemagglutinin could not be eluted from the cells by resuspending in phosphate-buffered saline (PBS), by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The receptor on mouse erythrocytes for the hemagglutinin was inactivated by trypsin, but not by neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin. The hemagglutinin was inactivated by trypsin, alpha-amylase, pepsin, DOC, KIO4, and ethylendiamine-tetraacetic acid (EDTA), but not by papain, beta-glucosidase, phospholipase C, neuraminidase, DTT, 2-ME, Tween-80, ethylether, chloroform, trichloro-trifluoroethane, beta-propiolactone and formalin, suggesting that the hemagglutinin active component involved glycoproteins. The hemagglutinin was stable at 37 degrees C for lower temperatures but not at 60 degrees C or higher. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 48,000 x g for 3 hr. In rate zonal centrifugation of the preparation on a sucrose density gradient, the hemagglutination (HA) activity showed a sharp peak at 1.22 g/ml coinciding with the peak of infectivity. The HA activity in the peak fraction seemed to be structually associated with virus particles. After fractionation of the virus by Nonidet P-40, the HA activity was found only in the fraction of the envelope material, indicating that the hemagglutinin is situated in the viral envelop.     

Evaluation of sprayed hypochlorous acid solutions for their virucidal activity against avian influenza virus through in vitro experiments

Hypochlorous acid (HOCl) solutions were evaluated for their virucidal ability against a low pathogenic avian influenza virus (AIV), H7N1. HOCl solutions containing 50, 100 and 200 ppm chlorine (pH 6) or their sprayed solutions (harvested in dishes placed at 1 or 30 cm distance between the spray nozzle and dish) were mixed with the virus with or without organic materials (5% fetal bovine serum: FBS). Under plain diluent conditions (without FBS), harvested solutions of HOCl after spraying could decrease the AIV titer by more than 1,000 times, to an undetectable level (< 2.5 log10TCID₅₀/ml) within 5 sec, with the exception of the 50 ppm solution harvested after spraying at the distance of 30 cm. Under the dirty conditions (in the presence of 5% FBS), they lost their virucidal activity. When HOCl solutions were sprayed directly on the virus on rayon sheets for 10 sec, the solutions of 100 and 200 ppm could inactivate AIV immediately after spraying, while 50 ppm solution required at least 3 min of contact time. In the indirect spray form, after 10 sec of spraying, the lids of the dishes were opened to expose the virus on rayon sheets to HOCl. In this form, the 200 ppm solution inactivated AIV within 10 min of contact, while 50 and 100 ppm could not inactivate it. These data suggest that HOCl can be used in spray form to inactivate AIV at the farm level.     

Enteric disease in specific-pathogen-free turkey poults inoculated with a small round turkey-origin enteric virus

Four- and 5-day-old specific-pathogen-free turkey poults were inoculated orally or by contact exposure to a small round turkey-origin enteric virus. At days 4 and 8 postinoculation (PI), the orally inoculated poults had significantly lower body weight gains than control poults. Poults at day 4 (orally inoculated) and 5 (contact-exposed) PI had watery droppings, dilated thin-walled ceca filled with yellow foamy fluid, catarrhal small intestinal secretions, pale intestinal serosa, and mild lymphocytic enteritis. In addition, at day 4 PI, poults were lymphopenic, had intracytoplasmic crystalline arrays of 17.1 +/- 1.1 nm viral particles in the jejunal villar enterocytes, and had an 18-to-24-nm virus in intestinal contents. Analysis of morphometric data revealed mild shortening of villi in the duodenum and elongation of crypts in the duodenum and ileum during the late stage of the syndrome (day 8 PI). These findings suggest that the 18-to-24-nm virus can produce an enteric disease syndrome and that the acute clinical manifestation of this syndrome is not the result of morphologic change such as intestinal villus atrophy. The definitive identity of this 18-to-24-nm virus is not known; however, based on size and intracytoplasmic arrays of virus, it is most probably an enterovirus.