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

Replication of Ibaraki virus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. The virus was resistant to ether, chloroform and deoxycholate, sensitive to trypsin, very labile at acidic pH but stable at pH 6.4 or higher, and was resistant to repeated freezing and thawing. The virus was readily inactivated at 56 C or higher, was fairly stable at 37 C, and very stable at 4 C, while it rapidly lost infectivity when stored frozen at —20 C. The virus was readily sedimented by centrifugation at 40 000Xg for 60 min. It readily passed through membrane filters of 200 mμ pore size, passed through 100 μfilters but only with some titer loss and did not through 50 mμ filters. In these tests, the bluetongue virus used as a control behaved in the same manner as Ibaraki virus. These findings provide additional evidence for the similarity of Ibaraki virus to bluetongue virus which had been previously demonstrated on the basis of seasonal incidence, symptomatology and pathology of the diseases caused by these viruses and the behavior of the viruses in cell cultures, embryonated eggs and laboratory animals. The present study, however, provided no evidence for any serological relation between these two viruses. More Information is needed to reach a final decision on the classification of Ibaraki virus, particularly regarding the morphology of the virion, the doublestrandedness of the viral RNA and other basic features.     

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.     

Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.     

Purification and some properties of Tephrosia symptomless virus

Tephrosia symptomless virus (TSV), isolated from Tephrosia villosa, is widely distributed in coastal districts of Kenya. The virus was readily transmitted by inoculation of sap, but not by Aphis craccivora or Apion sp. (Curculionidae) or through soil. Host range was very restricted and it infected only 10 of 70 species tested in one of nine plant families; susceptible species were confined to five genera within the Papilionaceae. The virus was cultured, propagated and assayed in soybean. TSV remained infective after 10 min at 85°C, 3 wk at 20°C and 26 wk at -12°C; crude infective sap of Glycine max retained infectivity when diluted 10^-6 but not 10^-7. Virus was purified from systemically infected soybean by clarifying sap extracted in 0.06 m phosphate buffer containing 0.001 m EDTA and 0.1% thioglycollic acid (pH 7.5) with equal volumes of 1:1 n-butanol/chloroform followed by two cycles of differential and one of sucrose density gradient centrifugation. Purified preparations contained c. 33 nm isometric particles. TSV contained RNA and one protein of molecular weight 1.53. 10⁶ and c. 42 000, respectively. Analytical centrifugation indicated a single component with a sedimentation coefficient (s.20, w) of 127 S; in Cs₂SO₄ and CsCl isopycnic gradients a single virus band formed; buoyant density in CsCl was 1.361. TSV was not related serologically to any of 44 viruses in nine plant virus groups but it resembled the tombusviruses and other ungrouped viruses such as carnation mottle in some of its properties.     

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.     

Citrus psorosis is probably caused by a bipartate ssRNA virus

Isolate 90-1-1 Concordia (Argentina) of the citrus psorosis agent was graft-transmitted to citrus and mechanically transmitted to Chenopodium quinoa, which was used as a local lesion assay host. Infected citrus and C. quinoa plant lesions were used as starting materials for the purification of the psorosis-associated agent.In extracts partially purified by differential centrifugation, infectivity was abolished by RNase treatment, even in 0.3 M NaCl, indicating that ssRNA is required for biological activity. The total loss of infectivity produced by proteinase K treatment and the decline in infectivity caused by phenol extraction indicated that protein may be essential for infectivity.When partially purified extracts were subjected to sucrose density gradient centrifugation, infectivity on C. quinoa from certain 2-fraction combinations was higher than expected, compared to the infectivity of the individual fractions. Therefore, infectivity was not associated with a single component but with the combination of at least two components which were distinguishable on sedimentation.The infectious material was present in the top and bottom zones of a sucrose gradient, which, on further purification by a second gradient and agarose gel electrophoresis, revealed the presence of a 50-kDa protein. This protein was absent in comparable gradient fractions from healthy plants, and therefore most likely represented the capsid protein of both the top and bottom sucrose gradient zone components.Taken together, these results led to the conclusion that the citrus-psorosis-associated virus (CPsAV) is a multipartite virus, containing ssRNA and a 50-kDa coat protein. In view of the information available to date, CPsAV seems to be very closely related to citrus ringspot virus described in Florida (USA) and the psoriasis agent in Spain.     

Differential Effect of Phleomycin on the Infectivity of Poliovirus and Poliovirus-Induced Ribonucleic Acids

The infectivity of intact poliovirus was not affected by exposure to the antibiotic phleomycin at concentrations as high as 200 mug/ml, whereas that of the singlestranded poliovirus ribonucleic acid (RNA) was inactivated to 99% by pretreatment of the RNA with phleomycin at a concentration of 2 mug/ml. The infectivity of double and multistranded RNA was 10 times less sensitive than that of singlestranded RNA to the action of this antibiotic. Preincubation of HeLa cells for 30 min with 10 to 50 mug of phleomycin reduced the sensitivity of the cells to infection by viral RNA and intact virus, indicating that phleomycin interferes with cellular functions necessary for virus replication. When phleomycin was added to cells at different times after infection with single- or double-stranded RNA, the highest inactivation of infective centers was observed immediately after infection. With time of incubation at 37 C, the infective centers became more resistant to the action of phleomycin.     

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.     

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Characterisation of rinderpest virus RNA and the action of actinomycin D on its replication

RNA extracted from purified rinderpest virus was characterised by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The predominant virion RNA species had a sedimentation constant of 46S and its estimated molecular weight was 4.8 × 10⁶ daltons. Consistently high amounts of UMP and AMP were detected. The melting-temperature profile of the virion RNA suggested absence of secondary structure. The effect of actionomycin D on the replication of rinderpest virus in Vero cells was studied by following the viral RNA synthesis using labelled uridine as well as by infectivity titration. The viral RNA synthesis was not affected until 12 h following infection and was inhibited thereafter between 18 and 48 h to an extent of 25% at 5 and 10 Μg levels of the drug. A 100 to 1000-fold reduction in the infectivity titres was observed in the presence of the drug. These results suggest that actinomycin D inhibits rinderpest viral RNA replication. Sedimentation analysis of viral RNA extracted from drug-treated cultures showed inhibition of the genome RNA of rinder-pest virus.     

Herpes Simplex Virus Products in Productive and Abortive Infection III. Differentiation of Infectious Virus Derived from Nucleus and Cytoplasm with Respect to Stability and Size

Approximately 67% of infectivity is associated with the nucleus 8 hr after productive infection of HEp-2 cells with herpes simplex virus. Comparison of nuclear and cytoplasmic infectious virus and macromolecular aggregates labeled with (3)H-thymidine or with (3)H-choline revealed the following. (i) Cytoplasmic infectious virus and macromolecular aggregates banded in CsCl at a density corresponding to enveloped nucleocapsids. The virus was relatively stable; there was only 50% loss of infectivity and only 16% of the virions became disaggregated. (ii) Nuclear macromolecular aggregates banded in CsCl solution at a density corresponding to unenveloped nucleocapsids and, moreover, both the infectious virus and aggregates were highly unstable. (iii) In sucrose density gradients, the nuclear macromolecular aggregates and infectivity sedimented as a single band and migrated more slowly than the corresponding cytoplasmic material. (iv) The infectivity of nuclear and cytoplasmic virus is readily inactivated by digestion with phospholipase C and with pronase. We conclude the following. (i) Cytoplasmic virus consists of enveloped nucleocapsids. (ii) Nuclear virus consists of nucleocapsids covered with lipid or partially enveloped. (iii) The molecular integrity of viral lipids is essential for infectivity. (iv) The envelope protects the nucleocapsid and accelerates adsorption to cells; it is not, however, inherently essential for infectivity.     

Specific Alterations of Coxsackievirus B3 Eluted from Hela Cells

After the attachment of radioactive coxsackievirus B3 to HeLa cells at 0 C and subsequent incubation at 37 C, 50 to 80% of attached virus radioactivity was eluted from the cells within 1 hr. Eluted virus had a buoyant density of 1.21 in a potassium tartrate gradient, sedimented more slowly than native virus in sucrose gradients, was resistant to ribonuclease, was unstable in CsCl centrifugation, and did not reattach to uninfected cells. Electrophoretic studies of sodium dodecyl sulfate-disrupted B3 virus in sodium dodecyl sulfate-polyacrylamide gels revealed four radioactive virus polypeptides (VP 1 to 4), of which the three largest migrated slightly faster than their poliovirus T1 counterparts. In contrast, electrophoretic analysis of eluted virus, after banding in a tartrate gradient or pelleting by centrifugation, showed the absence of the fastest migrating polypeptide, VP 4. VP 4 was recovered in the supernatant fluid when the eluted virions were removed by high-speed centrifugation. The results indicate that VP 4 is located at the surface of the native virion, and its dissociation from the capsid may represent the first specific alteration of the virion after virus-receptor interaction at the cell surface.     

Partial purification and evidence for multiple molecular forms of the scrapie agent.

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and detergent treatment with sodium deoxycholate yielded a fraction designated "P5" which was enriched for scrapie infectivity approximately 20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction P5 to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from 1.08 to 1.30 g/cm3. Parallel rate-zonal analysis showed that the infectivity was distributed over a range of particle sizes with s20.w values from approximately 40 S to greater than 500 S. Incubation of P5 at 37 or 80 degrees C, under conditions that disrupt ribosomes, dramatically altered the rate-zonal gradient profile of the agent. Under these conditions, the agent sedimented as particles with s20.w greater than 500 S. The apparent heterogeneity of the scrapie agent with respect to both size and density and its ability to shift from one form to another suggest that the agent may contain hydrophobic domains on its surface.     

Isolation and Characterization of Defective Interfering Particle of Newcastle Disease Virus

Newcastle disease virus grown in embryonated eggs was separated and purified by sucrose density gradient centrifugation into two distinct types of particles, B and T, the former being normal virus particles with high activities of hemagglutination, hemolysis, neuraminidase and infectivity, the latter being non-infectious virus particles with low activities of hemolysis and neuraminidase but high hemagglutination activity. B and T particles were shown to share a common antigen by immunodiffusion test. T particles were deficient in viral RNA, since they contained only 13s RNA in a small amount, whereas B particles possessed a large amount of 57s RNA and a small amount of 13s RNA. T particles interfered with the multiplication of normal Newcastle disease virus in primary cultures of chick embryo cells.     

Purification of the Kilham Rat Virus

A purification procedure is described for the isolation of Kilham rat virus (RV) from infected suckling hamster kidney and liver suspensions. The procedure involved a combination of sonic treatment, differential centrifugation, butanol-chloroform extraction, agar column flow diffusion, and potassium tartrate density gradient centrifugation. The purified virus retained its infectivity and was specifically neutralized by RV hyperimmune antiserum. Electron micrographs from the RV band (density 1.31 g/ml) showed numerous homogeneous particles approximately 22 mmu in diameter.     

Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.     

Some biological and physical properties of molluscum contagiosum virus propagated in cell culture.

Molluscum contagiosum virus propagated in FL cells of human amnion origin has a one-step growth cycle time of 12 to 14 h. The appearance and exponential increase of intracellular virus preceded the release of extracellular virus by approximately 2 h. Demonstration of comparable titers of extracellular and intracellular virus at the end of the replication cycle indicated that a substantial amount of virus remained associated with cells exhibiting cytopathogenic changes. Mean buoyant density values of virus in sucrose ranged from 1.275 to 1.278 g/cm3, but in CsCl the virus banded at densities at 1.325 to 1.340 and 1.261 to 1.281 g/cm3. Although virus infectivity was not affected by high concentrations of CsCl, it was found by polyacrylamide gel electrophoresis that the salt removed several nonglycosylated polypeptides with estimated molecular weights of 15,000 to 60,000. This suggested that the high-density band (1.325 to 1.340) may reflect the loss of these structural components. The half-life of virus infectivity was approximately 26.5 h at 26 degrees C and 11.2 h at 37 degrees C. Although the virus was rapidly inactivated at 50 degrees C, it could be stabilized at this temperature by the presence of 1.0 M MgCl2. Virus did not agglutinate newborn chick, adult chicken, or type "0" human erythrocytes. Virus infectivity was found to be sensitive to acid pH but resistant to treatment with diethyl ether or chloroform. The replication of molluscum virus in FL cells was not inhibited by 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or cytosine arabinonucleoside in noncytotoxic concentrations of 200 to 400 mug/ml, but greater than 99% reduction in the yield of herpes simplex virus or vaccinia virus in FL cells was obtained with 200 mug of these compounds per ml. Guanidinium chloride in concentrations of 100 to 200 mug/ml reduced molluscum virus yields by more than 99.9%.