Increase In Virus Concentration Research Articles

Purified vesicular stomatitis virus contains an enzyme activity that synthesizes cytidylyl (5'-3') guanosine 5'-triphosphate in vitro.

An enzyme activity that synthesizes cytidylyl (5'-3') guanosine 5'-triphosphate (pppGpC) in vitro has been identified in purified vesicular stomatitis virus. The activity is discernible after a lag period which is reduced in length with increasing virus concentration. The lag is eliminated by addition of pppGpC or ppGpC which are effective primers and stimulate dinucleotide synthesis linearly. The requirements of the reaction with respect to MgCl2, NaCl, and temperature are similar to those for viral mRNA synthesis in vitro. The activity, together with the viral L and NS proteins, is removed from virions by treatment with 0.8 M NaCl. The particulate fraction from infected cells that contains the transcribing subviral ribonucleoprotein particles also contains the enzyme activity. The corresponding fraction from uninfected cells does not, indicating that the activity is mediated by virus-specific proteins. Possible functions of the dinucleotide in the life cycle of the virus are discussed.

Respiratory infectivity of a recently isolated Egyptian strain of Rift Valley fever virus

The respiratory infectivity of a strain of Rift Valley fever virus isolated in Egypt (strain ZH-501) was compared with that of one isolate from Uganda (Entebbe strain) and two isolates from South Africa (strains SA-51 and SA-75). Studies were performed with ICR mice which were infected by exposure to infectious aerosols composed of particles with a mass median diameter of 0.96 micrometer. The respiratory median lethal doses for ZH-501, Entebbe, SA-51, and SA-75 were 2.2, 1.9, 2.6, and 1.9 log10 plaque-forming units, respectively. Although these values are statistically different, the biological implications of such differences seem unimportant. In an additional study of pathogenesis, a single group of mice was infected with 3.1 log10 plaque-forming units of ZH-501, and tissues were assayed sequentially through 96 h postinfection. Between 6 and 30 h, demonstration of an increasing virus concentration only in the lungs indicated that initial replication occurred there; however, determination of histopathological changes did not reveal evidence of pneumonia. Virus was isolated from the liver by 48 h, and the ultimate outcome of infection was a fulminating and fatal hepatic necrosis.

The significance of western equine encephalomyelitis viral infections in Aedes trivittatus (Diptera: Culicidae) in Iowa. I. Variation in susceptibility of Aedes trivittatus to experimental infection with three strains of western equine encephalomyelitis virus.

F1 pregnancy obtained from field-collected Aedes trivittatus were evaluated for susceptibility to infection with western equine encephalomyelitis (WEE) virus by intrathoracic inoculation and by oral imbibition of virus-blood suspensions through a membrane. Mosquitoes were uniformly susceptible to infection by intrathoracic inoculation of three strains of WEE virus, but minimum infective doses varied as much as 2,000 to 12,000-fold between strains by membrane feeding. Dose-response data obtained by membrane feeding also indicated that field strains of A. trivittatus were quite heterogeneous in their susceptibility to WEE virus since some individual mosquitoes could be infected by ingestion of low virus concentrations while others could not be infected by a 20,000-fold increase in virus concentration. Moreover, A. trivittatus showed a greater affinity for a WEE viral strain isolated from this species than for a WEE viral strain isolated from Culex tarsalis, even though the site, date of collection, and passage history of these isolates were identical. Field strains of A. trivittatus were relatively refractory to oral infection with WEE virus.

Poliovirus Movement During High Rate Land Filtration of Sewage Water

AbstractResearch with soil columns that are good models of a field ground water recharge system showed that most polioviruses are held near the soil surface. Secondary sewage effluent seeded with poliovirus type 1 (LSc) was filtered through 250‐cm columns packed with calcareous sand from an area in the Salt River bed that is used for ground water recharging of secondary sewage effluent. When the concentration of poliovirus added to the sewage water was increased from 0.9 × 102 to 2.6 × 104 PFU/ml, the number of viruses detected at each soil depth increased with the increasing virus concentration in the sewage water. However, the percentage of added viruses that remained at each depth was about the same for each concentration. The differences in the strength of the negative charge among members of a given viral population could account for the adsorption of some viruses near the soil surface while others move farther through the profile.Increasing the flow rate from 0.6 to 1.2 m/day caused a virus breakthrough of <1% of the added virus. However, 99% of the viruses still were removed from the infiltrating water at flow rates as high as 12 m/day. The velocity of water movement through the soil may be the single most important factor affecting the depth of virus penetration.

Practical Aspects of Survival and Destruction of Infectious Pancreatic Necrosis Virus

The survival of infectious pancreatic necrosis (IPN) virus under conditions of practical import and its susceptibility to destruction by two common disinfecting agents were examined. An indigenous strain of virus, shed to trough water at a concentration of 105 TCID50 per milliliter during an epizootic, lost 99% of its infectivity in fresh water at 4 C in 10–12 wk. There was residual infectivity after 24 wk. Survival was prolonged in sea water where loss of activity of an ATCC strain after 10 wk incubation at either 4 or 10 C was negligible. The loss in 5–6 mo was under 99%.IPN virus (107 TCID50/ml) survived drying in air at laboratory temperature and humidity for 5 but not for 6 wk. Under reduced humidity, residual infectivity was still evident after 8 wk.At a titer of 105.0 TCID50/ml, IPN virus was completely inactivated by exposure to 25 ppm available chlorine in 30 min. A concentration of 40 ppm available chlorine was required to inactivate 107.5 TCID50 of virus/ml in the same period of time. IPN virus, at a titer of 105.5 TCID50/ml, was totally destroyed by 30 ppm active iodine (Wescodyne) in 5 min. An increase in virus concentration, to 106.6 TCID50/ml increased the iodine requirement to 35 ppm to achieve the same result.The implications for salmonid culture of the persistence of IPN virus in an aqueous environment and its resistance to destructive agents are discussed.

Increase of wound tumor virus in leafhoppers as assayed on vector cell monolayers

Measurements of wound tumor virus (WTV) concentration in extracts from insect vectors ( Agallia constricta) were made by counting the foci of infection in monolayer cultures of the vector. Optimum conditions for performing such an assay procedure were determined. Brief exposure of insects to 50% ethanol and to certain antibiotics was necessary to avoid fungal and bacterial contamination in extracts prepared from the insects. The optimum pH value for the extraction of virus was 7.00 and for inoculation it was around 5.30 when a solution of 0.1 M histidine and 0.01 M MgCl 2 (His-MgCl 2) was used. When an incubation period of 40–46 hr at 30° was allowed, the optimal period of inoculation at 30° was 3 hr for insect extracts diluted to 10 −3.0 or higher. The focus counts of infected cells obtained by employing the optimal conditions for inoculation could be converted to absolute concentrations of virions by comparing the number of foci with those obtained from tumor extracts of known absolute virus concentration. After a 1-day acquisition period, virion concentrations in leafhoppers were determined for various days, starting from day 8, when the virus could be first assayed, to day 49. The virus concentration increased 50,000- to 100,000-fold between day 8 and day 25 after acquisition. Between day 23 and 28 the concentration reached a plateau level. After that the apparent concentration of virions was markedly affected by the extracting solution. The growth curve determined by focus assay on monolayers was similar to that determined previously by other methods except that the increase in virus concentration was followed over 1 to 1.5 additional log units. Extraction with His-MgCl 2 solution showed that the decline in virion concentration after the peak concentration, which was noticed in previous experiments, was almost entirely an artifact of extraction.

Effect of tomato spotted wilt virus on ribosomes, ribonucleic acids and fraction I protein in Nicotiana tabacum leaves

The effect of systemic infection with tomato spotted wilt virus on the concentration of ribosomes and on the synthesis of ribosomal RNA and Fraction I protein in tobacco leaves has been examined. Under the growth conditions used, virus entered the first systemically infected leaf 2 days after plants were inoculated and virus concentration, as measured by infectivity assay, increased to a maximum 5 days after inoculation and then declined. Systemic vein clearing appeared in this leaf 3 to 4 days after inoculation and, within a day, while the virus concentration was still increasing there was a rapid decline in the concentration of chloroplast (70 S) ribosomes to less than a quarter of that in comparable healthy leaves. Concentration of cytoplasmic (80 S) ribosomes declined at the same rate in both infected and healthy leaves. Two isolates of tomato spotted wilt virus in two different hosts gave similar results. Labelling of RNA and protein with radioactive precursors showed that the synthesis of chloroplast ribosomal RNA and Fraction I protein in the infected leaf was unimpaired on day 4 at the time of increase in virus concentration and decrease in chloroplast ribosomes, but their synthesis was inhibited 5 1 2 days after inoculation when the systemic leaves were developing vein necrosis, virus concentration had begun to decline and chloroplast ribosomes had become virtually undetectable. Under the same conditions, tobacco leaves systemically infected with tobacco mosaic virus showed a partial decline in the concentration of chloroplast ribosomes as systemic vein clearing appeared but there was no further loss as infection developed. The effect of infection with tobacco mosaic virus differes from tomato spotted wilt virus in that chloroplast ribosomal RNA and Fraction I protein syntheses were inhibited at the time of appearance of systemic symptoms.

Aphid inoculation of plants with viruses, viral ribonucleic acid, and hybrid virus.

While probing or feeding on leaves, Myzus persicae produced wounds that resulted in lesions when virus was applied later. Lesions were also produced on leaves covered with tobacco mosaic virus (TMV), tobacco mosaic virus ribonucleic acid (TMV-RNA), brome mosaic virus (BMV), or hybrid BMV (BMV-protein/TMV-RNA) when these leaves were subsequently exposed to aphids. With TMV the number of lesions increased with increasing virus concentration. Keeping plants in the dark reduced the number of lesions produced by aphids, but increased those produced by mechanical inoculation. Nicotiana glutinosa was more susceptible to inoculation of TMV by aphids than was N. tabacum var. Xanthi nc. It is concluded that infectible sites produced by mechanical means differ from those produced by aphids.Aphids could not transmit TMV-RNA, BMV, BMV-RNA, or hybrid BMV from a virus-covered leaf to a healthy leaf. However, BMV and TMV could be recovered from the mouthparts of aphids which had fed or probed on virus-covered leaves.

On the mechanism of persistence and distribution of barley yellow dwarf virus in an aphid vector

Barley yellow dwarf virus (BYDV) was recovered, using infectivity bioassays, from the gut, hemolymph, and salivary glands but not from the brain (including ganglia) of aphids Macrosiphum avenae (Fabricius) that had an acquisition access period of 4 days. As a source of virus inoculum, salivary glands were the poorest and gut was the best. However, the virus was not detected in the gut of aphids that were injected with a massive dose of virus, an observation suggesting that it does not pass from the hemolymph into the gut. The precipitin ring test detected virus antigens in the gut and hemolymph but not in the salivary glands and brain of aphids given a 4-day acquisition access period. Viruslike particles were observed dispersed in the lumen of the gut of exposed aphids, but their identity as BYDV particles could not be established, although large aggregations of BYDV particles were readily identified in the phloem cells of infected oat leaves. The relative virus concentration (as indicated by the percentage transmission of injected insects) in the gut, when assayed at various times following a 6-hour acquisition access period, remained at about the same level between 6 and 24 hours and declined thereafter up to 96 hours. The virus apparently did not multiply in the gut. That the virus can accumulate in the gut was demonstrated by the increasing virus concentration in this organ with increasing acquisition access periods. BYDV could not be maintained in the aphids by serial passage of virus from insect to insect. The ability of aphids to transmit the virus declined following acquisition or injection of the virus into aphids. Also, the period of retention of inoculativity and transmission efficiency of aphids was dependent on the dose of virus ingested or injected. These results suggest that BYDV does not multiply in its vector M. avenae.

Respiration of tobacco leaves infected with different strains of potato virus X

A study was made of the effects on respiration by four strains of potato virus X varying in the severity of symptoms they produce on tobacco leaves. No effects on respiration rates were observed in either inoculated or systemically infected but symptomless leaves, even though there was a significant increase in virus concentration during the experimental period. However, a significant increase was detected in any leaves on which symptoms developed, this increase being directly correlated with the severity of symptom expression. At the same time a great increase in virus concentration was found. Measurement of respiration patterns of single local lesions showed that an increase in respiration occurred each time a chlorotic and necrotic ring formed in the tissues. The use of metabolic inhibitors indicated that metabolic controls are different in the leaves infected with latent or mild strains, as compared to those infected with the severe, necrotizing strains.

Initial and subsequent sites of aster yellows virus infection in a leafhopper vector

Aster yellows virus (AYV) was recovered from hemolymph, alimentary canal, salivary glands and ovaries, but not from Malpighian tubules, mycetomes, fat body, testes, or brain of viruliferous leafhoppers, Macrosteles fascifrons (Stål). The presence of virus in the tissues was determined by injecting their extracts into virus-free leafhoppers, then testing the latter singly for inoculativity on aster ( Callistephus chinensis Nees.) seedlings. The virus was first recovered from the alimentary canal immediately after a 3-day acquisition access period and from the hemolymph and salivary glands on day 6 after the start of acquisition. The virus concentration, as indicated by the percentage of injected insects that became inoculative, increased rapidly in the alimentary canal, reached a peak by day 9, but then declined sharply by day 23 and remained at the same low level up to day 30, the longest time tested. The virus concentration in the hemolymph also increased rapidly, reached a peak by day 12, but remained at the same high level up to day 30. In the salivary glands the virus concentration increased gradually, and reached a high level by day 30. The increase of virus concentration in salivary glands was correlated with the increase in transmission by the source leafhoppers. The results suggest that the alimentary canal of the leafhopper is the initial site of virus multiplication, that hemocytes may be the main sites of virus multiplication, and that AYV must reach a certain concentration in the salivary glands before an insect can become inoculative.

THE INCREASE IN CONCENTRATION OF TWO STRAINS OF POTATO VIRUS X IN FLOATING LEAF DISCS

Increases in concentration of two antigenically similar strains (ringspot and yellow) of potato virus X (PVX) in Nicotiana tabacum L. vars. Samsun and Havana 38 and in Nicotiana rustica L. were determined serologically and by local lesion assay. In discs of inoculated leaf tissue floating on Hoagland's solution the rate of increase was greatest at 16° and 20 °C. In Samsun the optimum temperature for the increase in virus concentration was lower for the yellow strain than the ringspot strain. A close correlation between the assay procedures indicated that, as the temperature increased above the optimum for virus increase, either virus synthesis was being inhibited or virus was being denatured. No differences were found where 8- and 16-hour photoperiods were compared to determine their effect on virus increase at 20 °C. Virus concentrations in Samsun and Havana 38 were similar when determined under similar conditions. Concentrations in N. rustica were appreciably higher. In both Nicotiana species the concentration of the yellow strain at any time after inoculation was lower than that of the ringspot strain.

Studies on size of lesions of tobacco mosaic virus on Pinto bean

Strains U1 and U2 of tobacco mosaic virus produced both macroscopic (macro) and microscopic (micro) necrotic lesions on leaves of Phaseolus vulgaris L. var. Pinto. The lesions were recognizable also as starch lesions on leaves bleached with alcohol and stained with iodine. On green leaves, lesions of the two strains were distinguishable by color and size. Infectivity tests showed that virus from macro and micro lesions was biologically indistinguishable. In addition, the population distribution patterns of lesion size indicated that macro and micro lesions together constituted a unimodal, positively skew population. Therefore (1) the division of the lesion population into macro and micro lesions was an arbitrary division of lesions into two size groups; (2) the ratio of numbers of macro to starch lesions (at a magnification of 10×) provided a measure of lesion size; and (3) counts of starch lesions provided a more accurate measure of numbers of infections than counts of macro lesions. The ratio of numbers of macro lesions to numbers of starch lesions ranged from 45% to 75% for strain U1 and from 1% to 20% for strain U2. Size of lesions of strains U1 and U2 was increased by 80% and 76%, respectively, when ribonucleic acid rather than complete virus was used as inoculum. Size of lesions of strain U1 was increased with increase in virus concentration; the rate of increase in the ratio of macro to starch lesions per doubling of virus concentration was approximately 1%. For strain U1 there was heterogeneity in respect to lesion size between leaves of the same or different age and between areas within leaves.

Die Bildung der infektiösen Ribonucleinsäure während der Vermehrung des Tabakmosaikvirus

Young plants of Nicotiana tabacum (Var. Samsun) were infected with tobacco mosaic virus and kept at a constant temperature between 23 and 27°C and at constant illumination. The virus concentration was determined by bioassay on Nicotiana glutinosa. An exponential increase in virus concentration occurred 20-30 hours after infection. This latent period is significantly shorter after infection with virus ribonucleic acid. Probably the nucleic acid has to be liberated from the nucleoprotein before multiplication can start. The formation and multiplication of free virus ribonucleic acid could be demonstrated earlier than the formation of the complete virus. Infectious nucleic acid was measured by direct extraction of the plants with phenol. Nucleic acid included in the virus was determined after degradation of the free ribonucleic acid by incubation at 37°C and subsequent phenol extraction. The amount of free ribonucleic acid reaches a maximum 40 hours after infection and decreases afterwards to the extent as the virus bound ribonucleic acid increases. A general hypothesis for the biosynthesis of tobacco mosaic virus is given.

The Latent Period after Infection with Tobacco Mosaic Virus and Virus Nucleic Acid

THE process of infection by plant viruses is not so well known as that for animal viruses and bacterio-phages. The main reasons are that tissue cultures are not yet available in which all cells can be uniformly infected and that the methods for determination of the activity are not sensitive enough. Experiments of this kind have been carried out with total plants or pieces of leaves. Siegel et al. 1 studied the early events of infection with tobacco mosaic virus by an indirect method, measuring the ultra-violet sensitivity of infective centres in Nicotiana glutinosa. An increase in ultra-violet resistance occurred 5 hr. after infection at 20° C. This time can be considered as the lag phase. Other results to demonstrate the increase of virus concentration directly are obscured by the fact that the adsorbed virus which has not penetrated and therefore does not participate in the infection is not removed from the surface of the leaves2; further, the methods of determination were not sensitive enough3.

Onderzoekingen over een virusremstof voorkomend in Dianthus cariophyllus L. Werkingsspectrum, remmingsmechanisme en aard

An inhibitor of carnation proved to inhibit 14 very different viruses when tested on a range of 20 plant species. No viruses insensitive to the inhibitor were encountered. The degree of inhibition was for all viruses dependent on the plant species used as a test plant. The investigated plants could be distinguished into species very sensitive and hardly sensitive to inhibitor. The hardly sensitive ones all belonged to the order Centrospermae. The differences in sensitivity were quantitative and qualitative. The influence on contamination was expressed as a percentage activity. A decrease in the inhibitor's concentration hiw a stronger effect on the percentage activity than an increase in virus concentration of the same order. For various reasons it was considered improbable that inhibition would result from a reaction between virus and inhibitor in a liquid medium. Their mutual independence in vitro could be confirmed by ultracentrifuging sap from carnations with mosaic disease. The feasibility was also established of inhibitor acting through the test plant. Both infectious ribonucleic acid (RNA) of tobacco mosaic virus and the intact virus on Nicotiana glutinosa were inhibited by carnation inhibitor. The inhibition was shown not to result from an interaction between the inhibitor and the proteinaceous exterior of the virus. The virus-inhibiting activity of the inhibitor was finally interpreted on the basis of the 'receptor theory' of viral infection as a blocking of the sites near the leaf surface necessary for successful viral infection. By ultracentrifuging the molecular weight of the inhibitor could be estimated as at least 10.000. The inhibitor could also be isolated and some of its physical and chemical qualities could be established.