Bovine Diarrhea Virus

Virus strain No. 12, one of the new isolates from Japanese cattle described previously, was studied for its physicochemical properties. The new isolate was shown to be very small in size by centrifugation and filtration, being filtrable through Millipore filters of 50 mμ pore size. It appears to be an RNA virus as its replication was not inhibited by 5-iodo-2′-deoxyuridine. The virus was readily inactivated by ether and deoxycholate, and partially by trypsin; was labile at pH 3, not stabilized by 1 m MgCl₂ at 50 C, was inactivated by ultraviolet, and withstood repeated freeze-thawing. Further it was readily inactivated at 56 C but more slowly at 37 C, and was stable at lower temperatures. These findings support the identification of the isolated virus as the bovine diarrhea (BD) virus. The properties of BD virus, i.e. size, type of nucleic acid, ether, chloroform and deoxycholate sensitivities, and acid lability, appear to be similar to those of arboviruses. The trypsin sensitivity of BD virus is similar to the B group of arboviruses, which, unlike the A group, sensitive to trypsin. For the classification of BD virus as well as hog cholera virus, which is closely related, further elucidation of properties, fine structure of the virion, etc., is needed.     

Bovine Epizootic Fever

It was found that the ruins of Bovine Epizootic Fever appeared to be RNA as its replication was not inhibited by 5-iodo-2′-deoxyuridine. It had a buoyant density of 1.196, was not filterable through membrane filter (Millipore Filter Corp. Mass.) of 100 mμ pore size, and was sensitive to ether, chloroform and deoxycholate, was inactivated by trypsin and ultraviolet irradiation, precipitated by protamine sulfate, readily inactivated at pH 3.0, fairly labile at 56 C but readily preserved at —80 C, not stabilized at 50 C by I M MgCl₂, and resisted repeated freeze-thawing. The virus appears not to require a DNA-depcndent RNA synthesis in the host cell for its replication, as chromomycin A3 did not inhibit its replication. Sucrose density gradient centrifugation was unsuitable for purification because of a very poor recovery of infectivity. CsCl equilibrium density gradient centrifugation was successfully used for this purpose, and electron microscopy of the resulting fractions by phosphotungstic negative staining technique revealed virus-like bullet-shape particles, about 140 mμ in length and 80 mμ across, in the fraction of peak infectivity titer. The particle is probably the virion of the virus. These findings suggest relation of the virus to Rhabdoviruses [6], but further studies are necessary. The physicochemical properties of the virus provide additional evidence for similarity of the virus to bovine ephemeral fever virus, and emphasizes the desirability of further detailed comparative studies to decide whether they are one and the same or merely very closely related.     

Ibaraki Virus,an Agent of Epizootic Disease of Cattle Resembling Bluetongue

Ibaraki virus multiplied and induced cytopathic effects in primary cell cultures of bovine, sheep and hamster kidney and chick embryo, and cultures of BHK21-WI2 cells of baby hamster kidney origin and mouse fibroblastic L cells, but did not in primary cultures of horse and swine kidney cells and HeLa cell cultures. The virus was readily passaged serially in 4 to 5-day-old eggs using the yolk sac inoculation and incubation at 33.5 C. The viral growth was better in eggs incubated at 33.5 C than 37 C, and in younger eggs, with high yields in yolk, yolk sac and embryo. The virus was passaged serially in newborn mice by the intracerebral route. The virus multiplied in the brain of mice of any age, but younger mice supported better viral growth and developed encephalitis. As the age of mice increased, the morbidity and mortality became lower, no deaths being observed in 2 to 3-week-old mice. These observations in cell cultures, embryonated eggs and mice emphasize the similarity of Ibaraki virus to bluetongue virus. No evidence was obtained that young adult rabbits and weanling guinea pigs are susceptible to Ibaraki virus. The virus seemed to have little if any pathogenicity but infectivity of a low grade for sheep, while the virus is capable of inducing clinical illness, even severe in some instances, in cattle. This is in contrast to bluetongue virus which is highly pathogenic for sheep and much less so for cattle. Serial passages in embryonated eggs and suckling mice resulted in attenuation for cattle of Ibaraki virus.     

Isolation and properties of reovirus from cattle in an outbreak of acute respiratory disease.

A cytopathogenic virus was isolated in the primary culture of bovine kidney cells from a nasal swab of affected calves in an outbreak of acute respiratory disease in Japan in 1971. It agglutinated human type O erythrocytes and produced cytoplasmic inclusion bodies. Viral replication was inhibited by 5-iodo-2'-deoxyuridine, indicating that the viral nucleic acid was RNA. The virus was resistant to ether, chloroform, sodium deoxycholate, and acid, and passed readily through Sartorius' membrane filter 100 nm in pore size, but not through the filter 50 nm in pore size. Electron microscopy showed many spherical particles 60 approximately 75 nm in diameter with a double-layered capsid in a sample taken at a buoyant density of 1.34 produced by CaCl equilibrium centrifugation. The virus suspended in 1M MgCl2 solution was stable against heating at 50 degrees C for 30 minutes, but not against freezing at -20 degrees C for 60 minutes. The virus was resistant to, and increased in infectivity after, treatment with 0.063 approximately 1.0% trypsin. These properties were consistent with those established for the reoviruses. Most affected cattle showed a significant rise of antibody titer against reovirus and bovine respiratory syncytial virus, whereas only a few of them presented a serological evidence for recent infection with parainfluenza virus type 3, bovine adenovirus type 7, and bovine parovirus.     

Comparison of microporous filters for concentration of viruses from wastewater.

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Comparison of microporous filters for concentration of viruses from wastewater

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Antigenic and morphologic comparisons of Ibaraki and bluetongue viruses.

Bluetongue disease virus, type 10, and Ibaraki disease virus, which causes a bluetongue-like disease of cattle, were compared to determine whether they are the same or different viruses. They were similar in morphology, but neutralization tests, complement-fixation tests, and ferritin tagging indicated that they have antigenic differences. Therefore, they should be considered as different viruses. Two other viruses of this group, African horsesickness and equine encephalosis, were included to show that Ibaraki and bluetongue had developmental morphological features that could be used to differentiate them from the two equine viruses.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.     

Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.     

Processing and transport of environmental virus samples

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Processing and transport of environmental virus samples.

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.     

Experimental infection of Culicoides brevitarsis from south-east Queensland with three serotypes of bluetongue virus

Laboratory-reared C. brevitarsis (biting midges) were fed on sheep which had been experimentally infected with bluetongue serotype 1 (CSIRO 156), bluetongue serotype 20 (CSIRO 19) or bluetongue serotype 21 (CSIRO 154), or on cattle experimentally infected with bluetongue serotype 20 (CSIRO 19). Approximately 77 000 C. brevitarsis were exposed to sheep and 9000 to cattle. The average percentage feeding on sheep was 54% and on cattle 47%. In attempts to transmit virus by bite 3360 C. brevitarsis which had fed on viraemic sheep were held for 11-15 days before exposure to susceptible sheep. Although 11% of these insects fed, transmission of virus from sheep to sheep was not demonstrated. Estimated infection rates of C. brevitarsis for each serotype from sheep and serotype 20 from cattle were similar at 0.4% or lower. These low infection rates are one of the factors which make it unlikely that C. brevitarsis could be an efficient vector of bluetongue viruses in sheep in the field.     

Mechanism of Resistance to Enteroviruses of Some Primate Cells in Tissue Culture

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.     

Broad-bean stain and true broad-bean mosaic viruses

Autumn-sown crops of broad beans (Vicia faba L.) in England often contain plants with some leaves characteristically distorted and with a chlorotic mosaic. From some of these plants true broad-bean mosaic virus was isolated in 1959 and 1960 but not in 1965 and 1966. From other plants a similar but distinct virus, which caused staining of the seeds and we call broad-bean stain virus, was isolated in 1960, 1965 and 1966. The two viruses were readily distinguished in serological tests, and in some test plants. Both were seed-borne, and spread in crops, but were not transmitted by several animal species tested as vectors. Both viruses have isometric particles about 25 mμ in diameter. Some of these particles contain about 35% ribonucleic acid, some about 26% and some of those of broad-bean stain virus contain none; these three types of particles had sedimentation coefficients of about 120–130 S, 100 S and 60 S respectively. The ribonucleic acid of each virus had molar base content of G 23%, A 26%, C 18% and U 32%. These two viruses are members of the cowpea mosaic group of plant viruses; broad-bean strain virus was serologically related to cowpea mosaic, F I, red-clover mottle, and squash mosaic viruses. The particles of all these viruses and of true broad-bean mosaic virus were similar in appearance, sedimentation behaviour, and nucleic acid content and composition. The nucleic acid of red-clover mottle virus had a molar base content of G 20%, A 29%, C 20%, U 30%.     

ELECTRON MICROSCOPY OF CYTOPLASMIC INCLUSIONS IN CELLS INFECTED WITH ROD-SHAPED VIRUSES

Thin sections of leaf tissue infected with 12 rod-shaped viruses varying from 180 mμ to 750 mμ in length were examined in the electron microscope. Neither intranuclear nor cytoplasmic inclusions occurred in healthy tissue. Intranuclear inclusions were observed only in material infected with tobacco etch virus. Several types of cytoplasmic inclusions were induced by the group of viruses varying in length from 730 mμ to 750 mμ; however, only one type of inclusion was common to all seven viruses of this group. It is proposed that this inclusion, which appears as a pin-wheel in cross section and as a bundle in longitudinal section, is diagnostic for infection with viruses of the potato Y group, i.e., rod-shaped viruses whose lengths vary from 730 mμ to 750 mμ.     

Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Simple method for the concentration of influenza virus from allantoic fluid on microporous filters.

Membrane filter adsorption-elution is an efficient method for concentration and partial purification of several types of viruses from various aqueous solutions. For efficient virus adsorption to negatively charged filters, the sample is adjusted to pH 3.5 and trivalent salts are added before filtration. Since influenza virus is sensitive to extremes in pH, it cannot be concentrated by ordinary filters. Zeta Plus filters, which have a net positive charge of up to 5 or 6, were evaluated for the concentration of influenza virus from infectious allantoic fluids. Influenza virus efficiently adsorbed to Zeta Plus filters at pH 6, and addition of salts was not necessary. Adsorbed virus was eluted in a small volume of 2% bovine serum albumin plus 1 M NaCl at pH 10. By this procedure, viruses in 100 ml of allantoic fluid were concentrated to a final volume of 8 ml, with an average recovery efficiency of 71.0%.