Maize rough dwarf virus (Reoviridae) in its planthopper vector Laodelphax striatellus in relation to vector infectivity

The infectivity of females of the planthopper vector Laodelphax striatellus given access to maize rough dwarf virus (MRDV) infected plants was assessed for up to 55 days from the end of the access period. A 3-day inoculation access period was used, and this avoided intermittent transmission. Maximum infectivity was reached c. 30 days after acquisition access and the proportion of transmitter insects then remained constant. There was no difference in the efficiency of female L. striatellus in acquiring MRDV as third instar nymphs or as adults when compared in transmission tests 24, 30, 35 and 40 days after access to the virus. ELISA tests for MRDV subviral particles (SVPs) discriminated between individual viruliferous and non-viruliferous insects from the 30th day after access. Of the viruliferous (ELISA positive) insects about 30% did not transmit MRDV and the proportion remained similar from 30 to 55 days after access. None of the non-transmitter insects tested in serial transfer transmission tests was positive in ELISA. The concentration of SVPs detected by ELISA in the transmitter hoppers continued to increase exponentially, even after maximum infectivity was reached.     

A study of the mode of transmission of maize streak virus by Cicadulina mbila using an enzyme-linked immunosorbent assay

Two batches of Cicadulina mbila were given two distinct acquisition access periods (AAP) (3 h and 50 h) on maize plants infected with maize streak virus (MSV). Infectivity assays on susceptible maize were carried out 1, 3, 10, 17, 26 and 35 days after the AAP. Transmission efficiency was significantly higher for C. mbila subjected to the 50-h AAP. At the same time as the infectivity assays, the amount of MSV in each leafhopper was determined by an indirect double antibody sandwich (IDAS) ELISA. There were more ELISA-positive insects after the 50-h AAP than after the 3-h AAP. In the group given a 3-h AAP, only 7% of the insects tested between day 1 and 35 were found to be positive by ELISA. In contrast, after the 50-h AAP, the majority of C. mbila were positive, yet a decrease in ELISA-positive insects was noticed from day 17 onwards. Using a calibration curve obtained with purified virus, as little as 0.15 ng of MSV per insect could be measured by the IDAS-ELISA. A mean value of 0.36 ng of MSV per C. mbila was found 3 days after the 50–h acquisition, whereas 14 days later there was only 0.20 ng of virus per insect. For comparison, when leafhoppers were kept on infected maize, they displayed substantial accumulation of MSV up to an average of 3.83 ng of MSV per insect after 35 days of continuous acquisition. The amount of virus per insect detected in females was usually greater than the amount detected in males. Our results suggest that MSV does not multiply in C. mbila and contribute to the understanding of the persistence of transmission efficiency in the absence of virus multiplication.     

The particle morphology and some other properties of chloris striate mosaic virus

The causal agent of Chloris striate mosaic disease appears to be a virus with polyhedral particles 18 nm in diameter usually occurring as paired structures about 18 times 30 nm in negatively stained preparations. These particles were detected in the nuclei of infected plants forming characteristic inclusions in all cells except those of the epidermis. Such particles were not detected in thin sections of viruliferous leaf hopper vectors (Nesoclutha pallida). Purified virus preparations were shown to be highly infective when assayed by feeding vector leaf hoppers through membranes and confining them on indicator plants. In particle morphology, chloris striate mosaic virus (CSMV) differs from other viruses of Gramineae in Australia but resembles maize streak virus isolated in Africa, which however is serologically unrelated.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

The transmission of European wheat striate mosaic virus by Javesella pellucida (Fabr.) injected with extracts of plants and plant-hoppers

Virus-free individuals of the plant-hopper Javesella pellucida (Fabr.) infected plants with European wheat striate mosaic virus (EWSMV) after being injected at 5° C. with extracts of either plants or hoppers, but extracts of hoppers provided a better inoculum. Hoppers were unable to infect plants until at least 8 days at 20–25° C. after they were injected, and nymphs fed on infected plants similarly required 8 days before they gave infective extracts. Few hoppers survived more than a week after injection with untreated extracts of hoppers or with material sedimented from them by centrifuging the extracts at 8000g, but 60–70% survived injection with purer virus preparations. Injection of the virus seemed harmless, because as many hoppers survived CO₂ anaesthesis + injection, whether or not they later infected plants, as survived anaesthesis without injection. Attempts to determine the properties of the virus in vitro gave inconsistent results, but virus from hoppers was still infective after 10 min. at 30° C, 36 hr. at 5° C, precipitation at pH 4.0, storage for several months at -15° C, or at a dilution equivalent to 0.0014 g. hopper/ml. The best extraction medium contained 0.2 M-Na₂HPO₄+ ascorbic acid + 0.01 M-DIECA at pH 7.0–7.3. In sucrose density-gradients, EWSMV sedimented more slowly than tobacco mosaic virus. No specific particle with which infectivity could be correlated was seen by electron microscopy.     

Transmission of rayado fino virus of maize (Zea mays) by Dalbulus tnaidis

Rayado fino virus (RFV) of maize (Zea mays) was transmitted by the leaf-hopper Dalbulus maidis in a manner characteristic of viruses that multiply in their insect vectors. Individual insects fed on infected plants transmitted the virus after incubation periods of 8–22 days; males had shorter incubation periods than females but died sooner. Insects retained infectivity for 1–20 days. Transmission by most insects was intermittent. Inoculativity by D. maidis decreased with time, but the virus was recovered from insects that had lost their ability to transmit. Extracts of plants infected with RFV and viruliferous insects were injected into healthy insects, which became viruli-ferous. Infectivity of the extracts was not affected by tetracycline hydrochloride (Achromycin). D. maidis was able to transmit simultaneously RFV and the corn stunt agent. Other than maize, Teosinte (Euchlaena mexicana) was the only plant susceptible to the virus, among a number of species of Gramineae tested.     

Use of Clq enzyme-linked immunosorbent assay to detect plant viruses and their serologically different strains

Clq was prepared from bovine serum using a simple method involving repeated dialysis at low ionic strength in the presence of chelating agents (yield c. 3 mg/100 ml serum). It was viable when stored at -18°C for up to 2 months, and at 4°C for at least 10 wk in a storage buffer containing 10% sucrose. When used in Clq ELISA this test was as sensitive as the direct double antibody sandwich form of ELISA (direct ELISA) in detecting purified potato virus Y (PVY), with a limit of detection in both methods of c. 15 ng/ml, and slightly more sensitive in detecting purified cocksfoot mild mosaic virus (CMMV), with limits of detection of c. 15 ng/ml and c. 15–60 ng/ml respectively. Using an antiserum to one strain of each virus, Clq ELISA readily detected strains of PVY, CMMV, Andean potato latent virus (APLV) and barley yellow dwarf virus (BYDV). This included detection of APLV-Hu by APLV-Caj antibodies and CMMV(G) by PMV(S) antibodies, neither of which system gives detection in direct ELISA. Clq ELISA was therefore less specific than direct ELISA in detecting serologically different virus strains. Virus detection by Clq ELISA was inhibited when sap of tobacco, Nicotiana clevelandii and Setaria italica was used at low dilution. Inhibition by N. clevelandii sap was alleviated by using increased concentrations of virus specific antibody to detect APLV and plum pox virus. Also, extracting APLV infective N. clevelandii or CMMV infective S. italica saps in a minimum of buffer, centrifuging at low speed and diluting the supernatant before testing, partially overcame the inhibition. The inhibitory substance(s) in sap may act by preventing the binding of Clq to virus-antibody aggregates. Sap of wheat, oat and barley did not appear to have an inhibitory effect and BYDV was readily detected in naturally infected field grown plants of these species.     

Effect of anaesthesia with carbon dioxide on vectoring ability and survival of the planthopper Laodelphax striatellus

This paper describes the effects of carbon dioxide anaesthesia on the ability of the planthopper Laodelphax striatellus to subsequently acquire and transmit maize rough dwarf reovirus (MRDV) and barley yellow striate mosaic rhabdovirus (BYSMV), both propagative in the vector. Anaesthesia reduced the acquisition of MRDV by about 45%, but did not affect acquisition of BYSMV. In serial transfer transmission tests, anaesthesia caused interruption of transmission of both MRDV and BYSMV in about 11% of infectious insects; the effect may be due to impaired ability to find the phloem. The effect was stronger on third instar nymphs than on adults. Transmission of MRDV was resumed 3 days after anaesthesia, but none of the insects that stopped transmitting BYSMV resumed it. The survival of insects which ceased to transmit BYSMV was significantly lower than that of hoppers that continued to transmit. No other effects on survival were detected. A possible interaction of carbon dioxide with BYSMV in L. striatellus is discussed, in the light of effects of carbon dioxide on Drosophila melanogaster infected by some rhabdoviruses.     

The distribution of poplar mosaic virus in hybrid poplars and virus detection by ELISA

Purified poplar mosaic virus (PMV) at a concentration of 8 ng/ml was readily detected by enzyme-linked immunosorbent assay (ELISA). Bioassay in Nicotiana megalosiphon was more sensitive (detecting 1–4 ng/ml) and latex flocculation less sensitive (c. 25 ng/ml) than ELISA assays. While the foliar sap of fresh, naturally-infected poplars (e.g. Populus. euramericana cv. Robusta) was not infective at dilutions greater than 2 . 10–², ELISA easily detected PMV antigen when sap was diluted 4 . 10–³ in buffer or when one part of infected tissue was triturated with 99 parts healthy leaf. Furthermore, although sap from poplar leaves stored at -20 °C for 6 months was not infective, PMV was still detectable in ELISA tests. PMV antigen in poplar leaves was not all pelleted after centrifugation for 2.5 h at 130 000 g yet parallel tests using unbuffered sap from systemically infected Nicotiana megalosiphon foliage showed that infectivity was restricted to the pellet. In poplar foliage, the concentration of PMV antigen was generally greatest where symptoms were most obvious; least antigen was detected in the overwintering leaves located at the bases of long shoots. In winter, when root and inner bark tissue in the trunk was an erratic source of PMV, the virus was readily detected in buds, the concentration being greatest in the bases, including the meristem, of terminal buds. Propagation from single node cuttings of P. euramericana cv. Regenerata allowed the selection of clones that consistently showed either ‘severe’ or ‘mild’ foliar symptoms. The associated virus isolates also infected another poplar clone causing symptoms characteristic of their source. ELISA consistently detected less PMV antigen in field-grown cv. Regenerata than in cv. Robusta foliage, but this was reversed when the associated virus isolates were propagated in Nicotiana glutinosa at 24 °C. During 6 yr, 21 out of 127 poplars at a site in Western England, became infected with PMV. By contrast, in Eastern England, none of 46 were infected. The aphids Pterocomma populea and Myzus persicae did not transmit PMV.     

Transmission by Laodelphax striatellus Fallen of Rice black‐streaked dwarf virus from Frozen Infected Rice Leaves to Healthy Plants of Rice and Maize

Rice black‐streaked dwarf virus (RBSDV) is transmitted naturally to important crops such as rice, maize, barley and wheat in a persistent manner by the planthoppers, Laodelphax striatellus, Unkanodes sapporona and Unkanodes albifascia. Insect vector transmission tests are the basis for identifying viral incidence, evaluating the resistance of varieties and selecting resistance sources for rice and maize breeding. A simple, rapid and reliable method is described by which virus‐free small brown planthoppers (L. striatellus) acquired RBSDV from frozen infected rice leaves and transmitted it to healthy rice and maize plants. After feeding on frozen infected rice leaves, the planthoppers were tested by RT‐PCR for the presence of virus after 10, 15, and 22 days, respectively. The percentages of RBSDV‐containing insects were 0, 25 and 71.43% of L. striatellus fed on frozen infected rice leaves compared to 0, 28.25 and 71.43% of L. striatellus fed on fresh infected rice leaves, respectively. In transmission tests, three of eight rice seedlings (37.5%) and four of eight maize seedlings (50%) were inoculated by the planthoppers that had fed previously on frozen leaves and had allowed a 22 days latent period and showed typical disease symptoms. As a positive control, four of eight rice seedlings (50%) and four of six maize seedlings (66.67%) became infected. All rice and maize plants expressing disease symptoms were identified as virus‐positive by RT‐PCR. These results indicated that the planthoppers acquired RBSDV from frozen infected leaves and transmitted the virus to healthy plants.     

Quantitative studies on the uptake and retention of potato leafroll virus by aphids in laboratory and field conditions

Enzyme-linked immunosorbent assay (ELISA) was adapted for the efficient detection and assay of potato leafroll virus (PLRV) in aphids. Best results were obtained when aphids were extracted in 0.05 M phosphate buffer, pH 7.0, and the extracts incubated at 37 °C for 1 h before starting the assay. Using batches of 20 green peach aphids (Myzus persicae), about 0.01 ng PLRV/aphid could be detected. The virus could also be detected in single aphids allowed a 1-day acquisition access period on infected potato leaves. The PLRV content of aphids depended on the age of potato source-plants and the position of source leaves on them. It increased with increase in acquisition access period up to 7 days but differed considerably between individual aphids. A maximum of 7 ng PLRV/aphid was recorded but aphids more usually accumulated about 0.2 ng PLRV per day. When aphids were allowed acquisition access periods of 1–3 days, and then caged singly on Physalis floridana seedlings for 3 days, the PLRV content of each aphid, measured subsequently, was not strongly correlated with the infection of P. floridana. The concentration of PLRV in leaf extracts differed only slightly when potato plants were kept at 15, 20, 25 or 30 °C for 1 or 2 wk, but the virus content of aphids kept on leaves at the different temperatures decreased with increase of temperature. PLRV was transmitted readily to P. floridana at all temperatures, but by a slightly smaller proportion of aphids, and after a longer latent period, at 15 °C than at 30 °C. The PLRV content of M. persicae fed on infected potato leaves decreased with increasing time after transfer to turnip (immune to PLRV). The decrease occurred in two phases, the first rapid and the second very slow. In the first phase the decrease was faster, briefer and greater at 25 and 30 °C than at 15 and 20 °C. No evidence was obtained that PLRV multiplies in M. persicae. These results are compatible with a model in which much of the PLRV in aphids during the second phase is in the haemocoele, and transmission is mainly limited by the rate of passage of virus particles from haemolymph to saliva. The potato aphid, Macrosiphum euphorbiae, transmitted PLRV much less efficiently than M. persicae. Its inefficiency as a vector could not be ascribed to failure to acquire or retain PLRV, or to the degradation of virus particles in the aphid. Probably only few PLRV particles pass from the haemolymph to saliva in this species. The virus content of M. euphorbiae collected from PLRV-infected potato plants in the field increased from early June to early July, and then decreased. PLRV was detected both in spring migrants collected from the plants and in summer migrants caught in yellow water-traps. PLRV was also detected in M. persicae collected from infected plants in July and August, and in trapped summer migrants, but their PLRV content was less than that of M. euphorbiae, and in some instances was too small for unequivocal detection.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

The use of enzyme-linked immunosorbent assay to detect, and discriminate between, small iridescent viruses

The enzyme-linked immunosorbent assay (ELISA) double antibody method provided an efficient method for detecting iridescent virus (type 22) in purified preparations and extracts of Galleria mellonella larvae; 10 ng of purified virus/ml were detected with confidence. The ELISA method discriminated between the five iridescent viruses tested.     

Confirmation of the transmission of barley yellow mosaic virus (BaYMV) by the fungus Polymyxa graminis

Resting spores (cystosori) of Polymyxa graminis, selected from roots of barley plants infected with barley yellow mosaic virus (BaYMV), were used to start mono-fungal sand cultures. Out of 20 attempts using over 800 cystosori, P. graminis became established in 12, and in two of these BaYMV symptoms also occurred. BaYMV was detected by ELISA in extracts of dried roots heavily infected with cystosori and in zoospores of P. graminis. Calculations suggested that, on average, each zoospore carried less than 100 virus particles. In two virus acquisition experiments, non-viruliferous isolates of P. graminis failed to acquire BaYMV from roots of mechanically-inoculated plants. In two further experiments, non-viruliferous isolates were grown on rooted tillers produced from healthy plants and those infected with BaYMV by either vector or mechanical inoculation. Zoospores and cystosori of P. graminis subsequently transmitted the virus, but only from plants where it had been introduced by the vector. Repeated mechanical transmission appeared to have selected a strain of virus that could not be acquired and/or transmitted by the vector. The results provide convincing evidence that P. graminis is a vector of BaYMV but suggest that, in natural populations, only a small proportion of spores may be viruliferous.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。     

Ectopic expression of the spike protein of Rice Ragged Stunt Oryzavirus in transgenic rice plants inhibits transmission of the virus to insects

Rice ragged stunt oryzavirus (RRSV) replicates in both its insect vector, Nilaparvata lugens, and its plant host, rice, and has a complex multi-component particle bearing spikes on its outer surface. Transgenic rice lines expressing the 39 kDa spike protein showed good resistance to infection by RRSV. Furthermore, N. lugens fed on these plants prior to feeding on RRSV-infected plants were significantly protected against RRSV infection. The viral titre in insects initially fed on transgenic plants and then on RRSV-infected plants was inversely proportional to the levels of the 39 kDa protein expressed in the transgenic plants. This suggests that the 39 kDa protein interferes with the interaction between the intact virus particles and insect cell receptors and that the spike protein of RRSV contributes to vector specificity. This approach would probably be a more environment-friendly and sustainable method of virus control than by actual eradication of insect vectors.     

Rice Ragged Stunt Oryzavirus: role of the viral spike protein in transmission by the insect vector

A 39 kDa protein, known as the viral spike protein or one of the protein components forming the viral spike, encoded by genomic segment 9 (S9) of Rice Ragged Stunt Oryzavirus (RRSV) was obtained by enzymatic cleavage of a fusion protein expressed by S9 cDNA in bacteria with proteinase factor Xa. The feeding of an insect vector — the rice brown planthopper (Nilaparvata lugens) on purified expressed 39 kDa protein before the inoculation of the insects on diseased rice plants could completely inhibit the vector transmission ability of the insect. The presence of a 32 kDa insect cell membrane protein which could bind to 39 kDa viral spike protein indicated that the inhibition might be resulted from the competition in the interactions of 39 kDa protein and intact virus with the virus receptors on the insect cells. These results suggest that the spike proteins of the plant reoviruses are essential for the virus infection in the interactions of virus, insect vectors and host plants. These results are also useful in the practical applications.