Low Virus Concentrations Research Articles

Coxsackievirus B4 infection of the mouse pancreas: the role of natural killer cells in the control of virus replication and resistance to infection.

The role of natural killer (NK) cells in the early immune response to a pancreatropic isolate of coxsackievirus B4 (CVB4) was investigated in a murine model of pancreatitis. Endogenous (background) NK cell activity in fresh spleen effector cells from eight mouse strains was compared with virus-augmented NK cell activity 4 days post-infection (p.i.). A significant virus-induced increase (P less than or equal to 0.003) in NK cell activity was seen in seven of eight infected mouse strains, when virus titres in the pancreas were beginning to fall. Lesions in the exocrine pancreas were least extensive in the three strains with the highest endogenous NK cell activity. In C3H/HeJ mice that had been depleted of NK cells prior to infection with a low virus concentration, resistance to infection of the pancreas was completely abolished; myocarditis was also observed in one of these animals. Thus, NK cells may limit virus replication in the pancreas and play a role in resistance in C3H/HeJ mice. Virus-specific neutralizing antibody was not detected in the serum until 5 to 6 days p.i. in most strains and did not appear to influence pancreatic virus titres. It may be significant that CVB4 infection did not induce the expression of major histocompatibility complex (MHC) class I molecules on target acinar cells. With certain tumour cells, an inverse relationship between MHC class I expression and susceptibility to NK cell-mediated lysis is well documented.

Comparative epitope profiles of the particle proteins of whitefly‐transmitted geminiviruses from nine crop legumes in India

SummaryGeminiviruses associated with yellow or golden mosaic diseases of legume crops in two regions of India were compared by testing their reactivity with 27 monoclonal antibodies (MAbs) prepared to the particles of African cassava mosaic (ACMV) or Indian cassava mosaic (ICMV) viruses. The viruses fell into two main groups. Group 1 comprised isolates of dolichos yellow mosaic virus; these reacted with three or four ACMV MAbs and four ICMV MAbs. Group 2 comprised isolates of horsegram yellow mosaic virus, together with isolates from blackgram, cowpea, French bean, pigeonpea, soybean, Indigofera hirsuta and probably also isolates from mungbean. These reacted with three or four ACMV MAbs but with few or no ICMV MAbs. Isolates within each group differed slightly in epitope profile, depending on the source species (Group 2) or geographical origin (Groups 1 and 2). Isolates from lima bean resembled those in Group 2 but had some antigenic differences, and their status is uncertain. The poor detectability of geminivirus isolates in mungbean may reflect a low virus concentration in this species.

Barley Yellow Dwarf Virus Resistance in Triticum aestivum×Leymus angustus Hybrids

AbstractThe enzyme‐linked immunosorbent assay (ELISA) was used to determine the concentration of barley yellow dwarf virus (BYDV) in leaves of spring wheat Triticum aestivum L. cv. ‘Fukuhokomugi’, Leymus angustus (Trin.) Pilger (Altai wild rye) and their F1 hybrids inoculated at the 2‐ to 3‐leaf stage. The BYDV isolate “Cloutier” (serotype PAV) was used. The results showed that L. angustus confers immunity or strong resistance to the virus. Significant multiplication of the virus occurred in ‘Fukuhokomugi’ which expressed an intermediate level of tolerance to the disease under field conditions. The F1 haploid hybrids of ‘Fukuhokomugi’×L. angustus expressed a level of resistance almost equal to that of their wild parent, with a low concentration of virus appearing 16 days after inoculation and no more afterwards. The discovery of BYDV resistance in L. angustus and the expression of this character in the F1 hybrids provide new opportunities for the enlargement of the gene pool for BYDV resistance in wheat.

Acquisition of tomato yellow leaf curl virus by the whitefly Bemisia tabaci

Tomato yellow leaf curl virus (TYLCV) genomic DNA can be detected by Southern blot analysis in nucleic acid extracted from a single whitefly. Acquisition of TYLCV by individual whiteflies in relation to the length of the access period, the virus concentration in, and the developmental stage of plant tissues was studied. The frequency of TYLCV detection increased with the length of the access-period; DNA was detected in 15% of whiteflies tested after a period of access to infected tissue of 30 min, regardless of whether it had a high or a low virus content (5 ng or 0.05 ng TYLCV DNA/micrograms plant chromosomal DNA), and in all insects tested after an 8 h period of access to all the plants. Those insects which had access to the youngest leaves of source plants, which have a high virus content, acquired detectable TYLCV DNA within 2 h. Insects which had access to a tissue for the same period acquired variable amounts of TYLCV DNA; insects feeding on plants with a low virus concentration acquired amounts of viral DNA comparable to those acquired by insects feeding on plants containing a 100-fold greater concentration of virus. Viruliferous insects retained TYLCV DNA for at least 13 days when placed on uninfected tomato plants. In these tests, whitefly could not acquire more than 600 million virus genomes (1 ng viral DNA), suggesting the existence of factors controlling the number of virions present in an insect.

Regeneration of healthy banana plantlets from banana bunchy top virus‐infected tissues cultured at high temperature

Exposure of banana bunchy top virus (BBTV)‐infected plantlets of Cavendish banana to 40°C for 16 h daily for periods of up to 5 weeks did not free them from BBTV. However, BBTV was less readily transmitted by aphids from treated plants than from untreated controls. The same heat treatment applied for up to 12 weeks also failed to eliminate the virus from BBTV‐infected tissue. When similar BBTV‐infected tissue was cultured for 3 months at 35°C some of the buds started to produce healthy looking plantlets. Five out of 11 cultures produced healthy‐looking plantlets in 6 months. Crude extracts from the leaves of these plantlets did not react with a monoclonal antibody prepared against BBTV. When heat‐treated, healthy‐looking plantlets were inoculated with viruliferous aphids all plants developed bunchy top symptoms indicating that they were not resistant to BBTV. The results suggest that the uneven distribution and low concentration of virus after treatment at high temperature leads to BBTV‐free primordial cells which in turn may develop into healthy plantlets.

Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs

Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

Potentiation of Platelet Responses In Vitro by Feline Infectious Peritonitis Virus

The effect of feline infectious peritonitis virus (FIPV) on platelet aggregation and 14C-serotonin release induced by threshold levels of four agonists (adenosine diphosphate [ADP], collagen, arachidonic acid, and epinephrine) was examined in vitro in ten specific-pathogen-free cats. Purified suspensions of FIPV added to stirred platelet suspensions (virus to platelet ratio equal to 1:320) 1 minute prior to the addition of agonist potentiated the ADP-induced aggregation response by greater than 100% in seven cats. Platelet 14C-serotonin release was increased by greater than 100% in four cats. Collagen-induced platelet aggregation was enhanced in ten cats while collagen-induced 14C-serotonin release was enhanced in eight cats. Potentiation of arachidonic acid-induced platelet aggregation was observed in three cats, two of which demonstrated enhanced platelet 14C-serotonin release. Although epinephrine-induced platelet aggregation was enhanced in five cats, the samples displayed only fine microaggregates. Enhanced 14C-serotonin release from platelets in response to epinephrine was not demonstrated. Interaction with the outer platelet membrane and internalization of viral particles within the surface-connected open canalicular system were demonstrated by electron microscopy within 5 minutes of the addition of virus to platelet suspensions with or without added agonists. Decreasing the virus concentration by ten- or one hundred-fold abolished the potentiating effect observed previously, while increasing the concentration tenfold resulted in direct platelet activation in the absence of agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Limits of Detection of Bluetongue Virus with Different Assay Systems

The sensitivity of different assay systems for detecting low concentrations of bluetongue virus (BTV) were compared. These assays included blind passage on baby hamster kidney (BHK-21) cells and on cattle pulmonary artery endothelial (CPAE) cells, immunoperoxidase staining of cells on multiwell slides, and cDNA/RNA hybridization of BTV infected cells. Nine serial 10-fold dilutions of a cell culture-adapted BTV serotype 11 were tested (each dilution was treated as a separate sample) in all assays. Visual inspection for cytopathic effects (CPE) during 3 passages in BHK-21 cells detected samples that contained greater than or equal to 3 plaque forming units (PFU)/ml of BTV. Evidence of CPE during 3 passages in CPAE cells detected samples that contained greater than or equal to 0.3 PFU/ml of BTV. A limit of detection (greater than or equal to 0.3 PFU/ml) was obtained faster by immunoperoxidase staining of BTV-inoculated CPAE cells on multiwell slides and incubated for 3 days. The cDNA/RNA hybridizations of CPAE and BHK-21 cells incubated for 2 or 3 days, respectively, with BTV dilution samples detected samples that contained greater than or equal to 30 PFU/ml. Of the assay systems examined, immunoperoxidase staining of CPAE cells on multiwell slides inoculated with cell culture-adapted BTV was the most sensitive and fastest assay for definitive virus identification.

ウイルスの培養耳管線毛運動に及ぼす影響

Clinical investigation suggests that viral infection is involved in middle ear diseases including otitis media with effusion. We studied experimentally the direct effect of influenza A virus on the ciliary activity in the eustachian tube. Guinea pigs with normal otoscopic examination were used. Thin mucosal materials including epithelial layer of the midportion of the eustachian tube were obtained from each animal. Each material was immersed in RPMI 1640 medium. The most active ciliated cell of each culture was selected to be examined for ciliary activity (beats/min) at an ambient temperature of 30°C. Then each medium was replaced by RPMI 1640 containing a variety of influenza A viruses, and time-course changes of the ciliary activity of cells whose baseline activity was previously determined, up to 48 hours. Some materials cultured in the same fresh RPMI 1640 containing no virus served as controls. In the medium containing low concentrations of virus, ciliated cells did not demonstrate a dramatic decrease of ciliary activity. However, ciliary activity was decreased dramatically in the presence of higher concentrations (3.3×106PFU/ml), and most cells stopped their beating activity, Cultures exposed to virus were perfused with fresh RPMI 1640 for 12 hours to remove the virus, but quick recovery of the ciliary activity was not observed during observation, up to 12 hours.

Accumulation of double-stranded RNAs 3 and 4 in the symptomless leaves and green areas of mosaic-affected leaves of tobacco plants infected with cucumber mosaic virus.

A comparative study on levels of viral infectivity, yield, double-stranded (ds) RNAs and coat protein was conducted for the symptomless leaves (S), green areas (G) and yellow areas (Y) of mosaic-affected leaves from tobacco plants inoculated with an isolate of cucumber mosaic virus (CMV) containing no satellite RNA. Levels of CMV infectivity in S and G were very low (1-2% of Y), which agreed with extremely low virus concentration in S and G (less than 1% of Y). However, total amounts of dsRNAs in S and G were relatively high (33-43% of Y). Gel electrophoresis showed that Y contained almost equal amounts of all four dsRNA species, while S and G contained equivalent amounts of dsRNAs 3 and 4 but no dsRNAs 1 and 2. Levels of coat protein in S and G were also very low (1-3% of Y). Throughout the growth of the symptomless leaves, the amounts of dsRNA species and coat protein remained virtually constant. Periodic detection of dsRNAs from newly developed leaves at the shoot tip revealed that the amounts of dsRNAs 3 and 4 were generally constant, whereas dsRNAs 1 and 2 were detectable only in the mosaic leaves appeared before and after the period in which the symptomless leaves developed.

EFFICACY OF MIXTURES OF MICROBIAL INSECTICIDES AND PERMETHRIN AGAINST THE CABBAGE LOOPER (LEPIDOPTERA: NOCTUIDAE) AND THE IMPORTED CABBAGEWORM (LEPIDOPTERA: PIERIDAE)

AbstractThe joint action of permethrin and Bacillus thuringiensis var. kurstaki Berliner (B.t.), Autographa californica (Speyer) nuclear polyhedrosis virus (ACNPV), or Artogeia (= Pieris) rapae (L.) granulosis virus (ARGV) fed as mixtures to larvae of Trichoplusia ni (Hübner) or A. rapae in laboratory bioassys differed with the host insect, the components, and the concentrations of the components. Permethrin interfered with the activity of ACNPV and ARGV in most of the mixtures, particularly at low concentrations of the latter virus but mortality by mixtures of mid-range (approximately LC50 if fed alone) concentrations of permethrin and of ACNPV exceeded the mortality expected by the components acting independently. Concentrations of permethrin in the middle of the dosage range enhanced the effect of B.t. against T. ni resulting in mortality that exceeded the expected mortality but mortality of A. rapae larvae fed most permethrin–B.t. mixtures was less than expected.

A model of the effect of codling moth granulosis virus on Cydia pomonella

SummaryA model was developed based on an initial ingestion phase, followed by a period of virus multiplication and host death, to describe the effects of codling moth granulosis virus (CpGV) concentration and time from initial infection on the percentage of codling moth larvae (Cydia pornonella) dying from virus infection in the laboratory. With modification, this model also described the effect of CpGV concentration on the reduction of damage to fruit by C. pomonella larvae in the field. In both cases, the effect of CpGV increased with the l/10th power of virus concentration.LT50 (time to death of 50% of larvae) was inversely related to virus concentration in the laboratory.The model predicts the threshold concentration for death of larvae, as well as LD50(C50 and LD90(C 90,) for all ages of larvae.The model also predicts that if the majority of larvae entered through the sides of apple fruits, rather than through the calyx or near the stalk (as they probably did in the field), then the efficacy of CpGV in reducing fruit damage would be lowered. This effect of site of entry is especially marked at low virus concentrations.

ELISA Detection of Odontoglossum Ringspot Virus in Mature Plants and Protocorms of Cymbidium Orchids: Potential Solutions to Problems of Sample Preparation Time and Low Virus Concentration

AbstractThe ability to detect Odontoglossum ringspot virus (ORSV) released from the cut surfaces of leaf discs by ELISA was examined. Results indicate that ORSV from leaf discs can be detected but that multiple discs are necessary to obtain reliable detection of low virus concentrations, ELISA of Cymbidium protocorms, known from immunosorbent electron microscopy to be infected with ORSV, frequently produced A435 values which would be considered either negative or marginally positive on the basis of commonly accepted statistical limits (i, e. 2 x mean, or mean + 3 x standard deviation). The comparison of paired samples, one of which had been pretteated with ORSV antiserum, improved the sensitivity of the test from 10 ng ml ‘’ of virus to 2 ng ml’.

Development and Reproduction of the English Grain Aphid (Homoptera: Aphididae) on Wheat Cultivars Infected with Barley Yellow Dwarf Virus

Interactions among the English grain aphid, Macrosiphum (Sitobion) avenae (F.), and three wheat ( Triticum aestivum L.) cultivars infected with two isolates of barley yellow dwarf virus (BYDV) were investigated under laboratory conditions by caging individual aphids on leaves of healthy ‘Caldwell,’ ‘Abe,’ and ‘Talento’ wheat plants or plants infected with BYDV (P-PAV and RPV-NY isolates). M. avenae feeding on infected wheat had a significantly shorter (P ≤ 0.05) developmental time, a greater fecundity, and a greater intrinsic rate of natural increase ( r m) than aphids feeding on noninfected plants. These results suggest that BYDV and M. avenae interact for their mutual benefit, resulting in a higher potential for population growth of the virus and the vector. The wheat cultivar ‘Caldwell’ was found in this and previous studies to have a degree of tolerance to BYDV as indicated by milder symptoms. Resistance to virus production also was detected for the P-PA V-infected ‘Caldwell’ wheat, which had significantly lower ELISA values (i.e., lower virus concentration) than ‘Abe’ wheat. M. avenae also had a significantly reduced fecundity on ‘Caldwell’ compared with that on ‘Abe’ and ‘Talento’ wheats infected with BYDV. No significant differences were observed in M. avenae fecundity on BYDV-free cultivars. The results suggest that infection with BYDV might improve the nutritional quality of wheat to M. avenae , resulting in changes in the fecundity of the vector. These changes were greater with the BYDV-sensitive ‘Abe’ and ‘Talento’ cultivars than with the tolerant ‘Caldwell’ wheat cultivar.

Use of high-speed rolling to detect respiratory syncytial virus in cell culture.

We examined the effect of motion on respiratory syncytial virus (RSV) growth in cell culture. Infected cultures were incubated stationary, rolling, or on an orbital shaker. Enzyme immunoassay (EIA) results for cultures infected with high concentrations of a laboratory strain of RSV were similar for all incubation conditions. However, cultures infected with low concentrations of virus and rolled at 96 rpm had a significantly greater mean EIA optical density (1.78 +/- 0.22) than cultures rolled at 2 rpm (1.42 +/- 0.08) (P less than 0.05). The mean EIA optical density of high-speed-rolled cultures was also significantly greater than for cultures on an orbital shaker (1.25 +/- 0.08) or for stationary cultures (0.21 +/- 0.17) (P less than 0.01). The amount of virus measured by EIA from cultures infected with clinical specimens was also found to be significantly greater at 96 rpm than for stationary cultures. Cultures infected with cell culture isolates were detected significantly earlier at 96 rpm than when stationary. We suggest that high-speed-rolling can be used to enhance the detection of RSV in clinical specimens, especially if the virus is present in low concentrations.

Transgenic Plants that Express the Coat Protein Genes of Tobacco Mosaic Virus or Alfalfa Mosaic Virus Interfere with Disease Development of Some Nonrelated Viruses

To determine whether these plants also are protected against other viruses, they were inoculated with low concentrations of potato virus X (PVX), potato virus Y (PVY), cucumber mosaic virus (CMV), alfalfa mosaic virus (AIMV), and the cowpea strain of TMV (Cc-TMV). A delay in disease development also was observed when transgenic tobacco plants expressing the CP gene of AIMV were infected with PVX and CMV, but not when they were infected with TMV-U 1 . The results of these experiments demonstrate that transgenic tobacco plants that express different CP genes have a low but significant degree of protection against other viruses

Sensitive detection of two plant viruses by enzyme-amplified elisa

Enzyme-amplified ELISA (enzyme-linked immunosorbent assay) increased the sensitivity of detection of citrus tristeza virus and papaya ringspot virus in plant sap by 25- and 125-fold, respectively, compared with direct double antibody sandwich ELISA. The advantages of the new substrate system for the detection of low concentrations of viruses in plants are discussed.

Studies on the Progression of Aleutian Disease in Mink

One hundred ninetyfive male ADV-negative mink, including 79 pairs of brothers, were followed in their response to natural ADV-infection caused by mating with ADV-positive females and under epidemic conditions. Special attention was drawn to the development of progressive versus non-progressive Aleutian disease. This was done by plasmaelectrophoresis, detection of antibodies to ADV, and finally by macroscopical examination of mink organs at pelting time. We found that the progression of Aleutian disease presumably is under some genetic influence. We also found indication of differences in the response to ADV depending on how the infection was introduced. Mating to positive females (low virus concentration) resulted in significantly higher proportion of non-progressive responders than infection under epidemic conditions (high virus concentration).

Reaction to the potato leafroll virus (PLRV) in diploid potatoes

Diploid parents with some resistance to PLRV, were intercrossed to give 3 families with 191 clones which were evaluated for reaction to PLRV and yielding ability. After inoculation with PLRV the clones could be separated into those: 1) resistant, 2) susceptible, 3) intolerant, reacting with low virus concentration, 4) tolerant and 5) intermediate in reaction. Both the ELISA test and the evaluation of external disease symptoms were necessary to separate the clones. No correlation was found between resistance to PLRV and tuber yielding ability.

Inhibition of mechanical transmission of virus by sap, and infection of Cucurbita ecuadorensis with papaya ringspot virus type W

Dilutions of Cucurbita ecuadorensis sap were mixed with dilutions of sap from C. maxima infected with papaya ringspot virus type W (PRV-W), and the mixtures mechanically inoculated to seedlings of C. pepo cv. Small Sugar or Cucumis sativus cv. Supermarket. Plants inoculated with C. maxima sap plus C. ecuadorensis sap (at 1/8 when C. pepo was the test plant, or 1/16 when C. sativus was the test plant) showed reduced infection compared with plants not receiving C. ecuadorensis sap. The reduction caused by C. ecuadorensis sap was greater at a lower virus concentration (1 g infected C. maxima/400 mL sodium-potassium phosphate buffer 0.1 mol/L) than at a higher virus concentration (1 g/25 mL). It was concluded that C. ecuadorensis sap contains an inhibitor of infection, that tissue derived from this species should be diluted at least 1 g/ 10 mL buffer before use in inoculations, and that C. pepo, which was 5 times more sensitive than C. sativus, should be used as the test seedling in reisolations. When C. ecuadorensis, C. maxima and the F1 (C. maxima x C. ecuadorensis) were inoculated on their cotyledons with a severe isolate of PRV-W, symptoms were expressed on at least 1 plant of each species and the F1, and the virus was reisolated in seedlings of C. pepo cv. Small Sugar from the uninoculated leaves of all plants. Severity of symptoms and the proportion of plants infected at reisolation were least for C. ecuadorensis, intermediate for the F1 and most for C. maxima. This is the first report of infection of C. ecuadorensis by PRV-W.