- Volume 40, Issue 2, 1978

This paper is a report of studies on Cotia virus; this had been first isolated in 1965 in Brazil and was subsequently shown to be a poxvirus.

Cotia virus grew in a wide range of cell cultures and on the chick chorioallantois (CAM). Its growth characteristics are similar to those of other poxviruses. Microscopy showed virus factories or type B inclusions appearing before infectious progeny virus could be demonstrated. Type A inclusions appeared later, after development of progeny virus; these were shown by electron microscopy to differ from the type A inclusions of cowpox and other poxviruses and they have been termed Cotia bodies. Immunofluorescent staining also showed ring structures which appeared before the development of Cotia bodies. The growth of Cotia virus in human embryo lung (HEL) cells was sensitive to inhibitors of DNA and protein synthesis but was resistant to a concentration of rifampicin which inhibited vaccinia virus. Sharing of antigens between the Cotia virus and vaccinia virus was shown by gel precipitation tests and immunofluorescent staining. There was no cross neutralization between Cotia virus and vaccinia virus nor did anti-Cotia sera neutralize representatives of other poxvirus groups.

African swine fever virus emerges from infected Vero cells either from areas of smooth cell surface or from microvilli. The two patterns may occur at different sites on the same cell and are unique for this virus. The scanning electron micrographs supplement regular thin section views.

Lymphocystis virus has been classified with the icosahedral cytoplasmic deoxyriboviruses (ICDVs) but a detailed structure of the virion has not yet been established. Virus from tumours of European plaice and flounders was allowed to decay and this revealed details of its structure. Freshly prepared virus has a hexagonal outline which breaks down to reveal surface subunits 4.5 nm in diam. and a spheroidal core with tubular material 13 nm in diam. inside it. This tubular material appears to have a periodicity suggesting a helical conformation and may be a deoxyribonucleoprotein. No triangular or pentagonal facets, as found with some other ICDVs, were seen. A possible construction of the virus is proposed.

Particles of cynosurus mottle virus (CyMV) purified from systemically infected oat leaves have a sedimentation coefficient of 113S, buoyant density of 1.399 g/ml in CsCl and 1.332 in Cs2SO4, diffusion coefficient of 0.130 × 10−10 m2/s and E 0.1% 260 of 7.3. Virus particles are isometric with a diameter of 27 to 29 nm in negatively stained preparations; the hydrodynamic diameter was calculated from the D 20, w as 32.8 nm. The particle mol. wt. was calculated as 6.86 × 106. CyMV particles contain 22.5% single-stranded RNA of mol. wt. approx. 1.5 × 106 and a molar nucleotide composition of: A, 24.2%; G, 24.3%; C, 26.2% and U, 25.3%; RNA purified by the phenol-SDS method was infectious. The melting profile of native CyMV-RNA was typical of a single stranded RNA with secondary structure; the hyperchromicity was 33% and the Tm 61 °C. The protein mol. wt. was 30500. The completed cryptogram for CyMV is R/1:1.5/22.5:S/S:S/Ve/Ap.

CyMV particles were degraded by 0.02 to 0.05% SDS at pH 7.0 into a component with a sedimentation coefficient of 83S and by 0.1% SDS into the RNA and protein constituents; virus dissociation was enhanced by EDTA and reduced by MgCl2. At pH 5.0, the virus was not affected by SDS concentrations of up to 8%. The virus was unstable in EDTA and NaCl at pH 8.25.

Western equine encephalitis virus, and RNA virus, and herpes simplex virus type 1, a DNA virus, were efficiently inactivated in less than 1 min by exposure to long-wave ultraviolet light (320 to 380 nm) in the presence of several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the light nor any of the drugs alone caused appreciable inactivation. The inactivation kinetics and dependence on light intensity and on different derivatives of psoralen were studied. The high solubility of a new aminomethyl psoralen derivative was found to be advantageous in the photochemical inactivation of the RNA virus, but was not in the case of the more easily inactivated DNA virus. Within its limited solubility range trimethylpsoralen was superior to its aminomethyl derivative on a molar basis for the inactivation of both types of viruses under most of the conditions studied.

The enzyme linked immunosorbent assay (ELISA) has been shown to be a specific and sensitive method to detect a nuclear polyhedrosis virus in Heliothis armigera larvae. 1 ng/ml of purified virus particle antigen could be detected. The presence of larval extracts did not interfere in the assay. The growth of the virus in Heliothis armigera larvae was analysed by ELISA and the conventional counting of virus polyhedra and this showed that ELISA was a superior detection method.

The formation of defective herpes simplex virus (HSV) in BSC-1 cells and the synthesis of defective virus DNA was studied. The fourth consecutive passage of undiluted virus yielded defective DNA that was 0.008 g/ml more dense than wild type (w.t.) virus DNA. The amount of defective DNA increased at passage 6 concomitantly with the decrease in infectious virus progeny. The synthesis of defective DNA was always accompanied by w.t. virus DNA synthesis. Defective DNA from both infected nuclei and defective virions had a mol. wt. of 100 × 106 and was linear as determined by electron microscopy. Electron microscopy of defective virus DNA at passage 6 revealed circular molecules varying in size in addition to linear DNA molecules with the length of intact virion DNA. The circular DNA molecules had contour lengths of 10, 5, 2.5 and less than 2.5 µm. The smallest circular DNA molecules had a contour length of 0.3 µm, possibly one virus gene. In addition, circular-linear DNA molecules were observed in which both the circular and the linear components varied in length. Most of these DNA molecules had circular components of either 2.5 or 5.0 µm, and linear components varying in length from less than 1 to 50 µm. Based on the present study, it is proposed that the S component of w.t. virus DNA is fragmented into small circular molecules that serve as templates for DNA synthesis, possibly by the rolling circle mechanism.

Calcium chloride (5 to 20 mm) potentiated interferon production induced by rIn:rCn in L929 mouse fibroblasts up to a thousand-fold. Higher concentrations of calcium (20 to 65 mm) mixed with rIn:rCn were associated with increased cytotoxicity and a more acidic medium, but were effective in enhancing interferon production if preparations were adjusted to a uniform pH. Although calcium increased cellular binding of 3H-rCn:rIn, only a partial correlation between binding and interferon production was observed.

Serological relationships between plant viruses within six different taxonomic groups were studied by means of the double antibody sandwich form of enzyme-linked immunosorbent assay (ELISA). This technique was very sensitive not only for the detection of low concentrations of viruses, but also for the differentiation of closely related antigens. With some viruses, e.g. Andean potato latent virus, the specificity of the test was so great that conjugates prepared to one strain failed to detect other serologically closely related strains. With other viruses and with host proteins the cross-reactivity was broader, but distant and even intermediate serological relationships between viruses were usually not detected. The great specificity of ELISA is mainly caused by the behaviour of the conjugated antibodies. The binding of conjugated antibodies was completely inhibited by simultaneously added native antibodies. Antibodies from very early bleedings were not suitable for ELISA.

The replication of human cytomegalovirus (CMV) and herpes simplex virus (HSV) was studied in three human embryo cell lines (CMV-Mj-HEL-1, CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-1) transformed in vitro by human CMV. Growth studies revealed that these cells were completely resistant to infection by CMV strains AD169 and Mj and partially resistant to HSV types 1 and 2. Neither virus DNA nor virus proteins were synthesized in the transformed cells infected with CMV AD169. The HSV production in CMV-transformed human embryo lung (HEL) cells was delayed when compared to the virus production in normal HEL cells and spread of HSV c.p.e. was slower in the transformed cells. The treatment of normal HEL cells with a crude extract of CMV-transformed HEL cells also resulted in inhibition of the spread of c.p.e. of HSV types 1 and 2. The inhibitory effect was not due to interferon since vesicular stomatitis virus replication was not affected and several experiments showed that it was not due to mycoplasma. The presence of virus inhibitor molecules in CMV-transformed cells absent in normal HEL cells is postulated.

Surface antigens of BHK-21 cells infected with HSV-1 or HSV-2 were radio-iodinated (125I) with lactoperoxidase, immune precipitated and analysed by polyacrylamide gel electrophoresis (PAGE). Experiments using antiserum to HSV-1 or HSV-2, absorbed with appropriate homotypic or heterotypic antigens, revealed that both type-specific (homotypic) and cross-reactive antibody combined with surface glycoproteins to form a single large radioactive peak. This peak, which constituted the major glycoprotein region (region a) observed in electropherograms, represented a range in mol. wt. from 115000 to 130000. Sensitization of cells to complement lysis, neutralization of infectious virus and immune precipitation of surface glycoproteins (region a) were found to be generally correlated properties of all the antibody preparations analysed, including antibody prepared specifically against region a antigens. These findings suggest a major immunological role for the surface glycoproteins migrating in PAGE region a.

Some properties of a small RNA virus isolated from Mansonia uniformis are described. The virus particles have a sedimentation coefficient of about 165S, buoyant density in CsCl of 1.33 g/ml and diameter of 28 nm. They contain a single-stranded RNA which probably lacks poly (A) but is infective. Four polypeptides with mol. wt. of 33, 30, 27 and about 7 × 103 are present in the virus particle.

The uptake of 3H-inositol into the pool of free inositol and its incorporation into the lipid phosphatidylinositol have been studied in various avian and mammalian cells infected by different viruses. In all the virus-cell systems investigated, virus infection results in a drastically reduced amount of free 3H-inositol about 3 to 5 h post-infection, demonstrable in the infected cells as compared to the mock-infected controls. In contrast, the incorporation of 3H-inositol into lipid can be enhanced, reduced, or not influenced at all, depending on the virus-cell system under observation.

Circular dichroism studies (CD) of turnip yellow mosaic virus (TYMV) nucleoprotein and of its isolated RNA and capsid revealed that: (i) the nucleic acid structure, which comprises a considerable amount of base pairing and/or stacking, remains essentially unchanged irrespective of whether the RNA is encapsidated or free; (ii) the secondary structure of the protein component is mainly accounted for by β- and irregular forms without appreciable amounts of α-helix; (iii) the interaction of capsid protein and RNA induces some conformational changes in the protein probably involving a decrease of β-structure and a perturbation of the microenvironment of some aromatic residues. The influence of temperature on the CD spectra of virus nucleoprotein, RNA and capsid was also investigated. The results are discussed in connection with particle stability.

Changes in enzymes and metabolites of the carbohydrate metabolism in skeletal muscles were studied in mice after intracerebral inoculation of dengue type 2 virus. It was noted that lactic dehydrogenase, aldolase, phosphogluco-isomerase, phosphoglucomutase, GO-T and GP-T activity were enhanced initially by two- to three-fold, reaching a peak on day 5. As the illness appeared in mice, all the enzyme activities were lowered and were about three times less in the paralytic stage on the 8th day as compared to controls. Fructose-1,6-diphosphatase activity was increased on the 4th and 5th days but decreased later. Acid phosphatase increased abruptly from the 6th day while alkaline phosphatase activity was irregular. Creatine increased on the 4th and 5th days but diminished later. Glycogen decreased from the beginning and was lowest on the 5th day, but the levels increased later and were maximum in paralysed muscles. On the other hand, lactic acid began accumulating in the muscles and was maximum on the 5th day, then declined. Dengue virus was detected in the muscles from the 2nd day but higher titres were seen from the 6th day. Changes similar to the preparalytic stage of mice may occur in human beings, causing myalgia.

The neutralization of feline leukaemia virus could be enhanced by performing the reaction in hypotonic medium. Absorption of antibody by virus specific antigens was also made more sensitive under these conditions.

Disodium, phosphonoacetate (PA), at concentrations of 50 to 200 µg/ml, which still allowed continued growth of the EB virus-transformed B95-8 cell line on a routine culture regimen, was able to inhibit the production of virus capsid antigen and of virus particles by these cells down to very low but finite levels which persisted despite prolonged treatment. Further experiments measured the effects of these same drug concentrations on the EB virus-induced in vitro transformation of foetal cord blood lymphocytes and on the colony forming ability of already established EB virus-transformed foetal cell lines; in both types of culture, doses of PA up to and including 50 µg/ml did not affect cell growth within the 8-week observation period, whereas doses of 100 µg/ml and above were increasingly inhibitory. The cell lines established by EB virus-induced transformation in the continual presence of PA at 50 to 150 µg/ml contained multiple copies of the virus genome per cell just as did the corresponding cell lines established in control medium. The results argue against the existence of any PA-sensitive event unique to the EB virus-induced transformation process.

The stability of the in vitro infectivity of the non-occluded virions (NOV) of the Autographa californica nuclear polyhedrosis virus (Ac-NPV) was tested. Exposure of Ac-NPV NOV to solutions ranging from pH 7.5 to 11.4 with molarities of 0.1 to 0.001 showed that infectivity was most stable in 0.001 m, pH 7.5, tris(hydroxymethyl)aminomethane (tris)-HCl buffer. Infectious NOV could be concentrated by precipitation with 50% saturated ammonium sulphate or 9% (w/w) polyethylene glycol 4000. Treatment of concentrated preparations with 1 m-urea did not affect infectivity or the centrifugal properties of the NOV. NOV were partially purified by centrifugation through a sucrose underlay.

Human cytomegaloviruses (HCMV) induce nuclear antigens as early as 1 h after infection as detected by immunofluorescence. Early antigen development occurs in the presence of DNA, RNA, and protein synthesis inhibitors and is dependent on intact viral DNA in the inoculum.

Mesophyll protoplasts isolated from plants of wheat, oat, maize (a local lesion host) and Japanese radish (normally a non-host) were efficiently infected with brome mosaic virus (BMV) without using poly-l-ornithine, and the optimum conditions for infection were very similar to those found using barley protoplasts. These results suggest that the host ranges of viruses can be extended by using protoplasts and that the conditions optimal for infection of protoplasts with BMV are determined mainly by the virus.