Sublethal effects of iridovirus disease in a mosquito

Recognition of the importance of debilitating effects of insect virus diseases is currently growing. Commonly observed effects of sublethal infection at the individual level include extended development times, reduced pupal and adult weights, and lowered fecundity. However, for the most part, sublethal infections are assumed to be present in survivors of an inoculum challenge, rather than demonstrated to be present by microscopy or molecular techniques. Invertebrate iridescent viruses are dsDNA viruses capable of causing disease with symptoms obvious to the naked eye, a “patent” infection, that is lethal. Furthermore, inapparent “covert” infections may occur that are non-lethal and which can only be detected using bioassay or molecular techniques. In this study, replication of Invertebrate iridescent virus 6 in Aedes aegypti larvae was demonstrated in the absence of patent disease. A sensitive insect bioassay (using Galleria mellonella) allowed the detection of covert infections, which were more common than patent infections. A concentration-response relationship was detected for the incidence of patent infections. Covert infections were up to 2 orders of magnitude commoner than patent infections, but the prevalence of covert infections did not appear to be related to virus inoculum concentration. Exposure of larvae to virus inoculum resulted in extended juvenile development times. A reduction in the mean and an increase in the variability of fecundity and adult progeny production was observed in females exposed to an inoculum challenge, although formal analysis was not possible. Males appeared capable of passing virus to uninfected females during the mating process. Covertly infected females were smaller and had shorter lifespans than control or virus-challenged females. A conservative estimate for the reduction in the net reproductive rate (R ₀) of such insects was calculated at slightly more than 20% relative to controls. Received: 5 October 1998 / Accepted: 13 February 1999     

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.     

Field trials withTipula iridescent virus againstTipula spp. larvae in grassland

Field trials withTipula iridescent virus (TIV) were carried out to determine whether the infection can be introduced into populations ofTipula spp. in grassland. The virus was introduced into plots in live and deadTipula oleracea L. larvae, in a bran bait and in sprayed aqueous suspensions. Trials were conducted at 1 site in 3 successive years and at 5 further sites in the 3rd year. Tipulid larval populations in the plots were sampled at intervals of approximately 2 months. The majority of sampled larvae were not iridescent and did not become iridescent when they were incubated at 20°C for 30 days. In plots where iridescent larvae were found they generally comprised between 1 and 17% of the tipulid population. The identity of the virus infecting these insects was confirmed by the latex agglutination test. The results suggest that all the treatments introduced the virus infection into one or more of the tipulid populations; they all did so, however, with low efficiencies.

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Résumé Des essais en parcelles avec le virus irisant deTipula (TIV) ont été effectués pour déterminer si l'on peut introduire l'infection dans des populations deTipula spp. en prairie. Le virus a été utilisé sous forme de larves vivantes ou mortes deTipula oleracea L., d'appat de son, et en suspensions aqueuses. L'expérimentation a été réalisée dans un emplacement pendant 3 ans successifs et dans 5 sites complémentaires pendant la 3^e année. L'échantillonnage des populations de larves de tipules a eu lieu tous les 2 mois. La majorité des larves récoltées n'etaient pas irisantes et elles ne le sont pas devenues après un élevage à 20°C pendant 30 jours. Dans les parcelles où l'on a trouvé des larves irisantes, celles-ci représentaient 1 à 17% de la population de tipules. L'identité du virus dans ces insectes a été confirmée par agglutination au latex. Les résultats suggèrent que tous les traitements ont introduit l'infection virale dans les populations de tipules mais avec une faible efficacité.

     

A Tipula paludosa population with a high incidence of two pathogens

The incidence of lethal parasites in the larvae of a Tipula paludosa population was monitored for two seasons. The proportions of larvae infected with Tipula iridescent virus (TIV) and a tachinid insect were similar to those in previously studied populations, whereas the proportions of larvae infected with Tipula nuclear polyhedrosis virus (NPV) and a spore-forming bacterium (SFB) were higher. Conservative estimates of mortality due to these four agents were 10.7% in 1977–1978 and 7.7% in 1978–1979. The mean population density and the proportion of SFB-infected larvae were lower in 1978–1979 than in 1977–1978, while the proportion of NPV-infected larvae was higher. In 1979 the proportion of NPV-infected larvae was positively correlated with population density, which was highest in the wettest part of the study area. In both seasons the proportion of SFB-infected larvae was negatively correlated with population density. Larvae infected with the NPV or the SFB became pallid at an advanced stage of infection, but, although infected larvae were found throughout the larval period, pallid larvae were only found in the later part. It is suggested that larvae become infected in an early instar, then the infections slowly develop throughout the remainder of the larval period. Five larvae were found with mixed infections; four were infected with the SFB and NPV, while the fifth was infected with the SFB and TIV.     

Patterns of covert infection by invertebrate pathogens: iridescent viruses of blackflies

Recently, it has been recognized that blackfly populations may host two forms of infection by iridescent viruses (IVs); a covert (inapparent, nonlethal) form which was common in springtime populations in the River Ystwyth, Wales, and a patent (obvious, lethal) form which was rare. This study aimed to investigate the changes in frequency of the two types of infection in blackfly populations over the reproductive period of the flies, April-September 1992. Blackfly larvae sampled from three different sites along the river were bioassayed for the presence of covert IV infection. Of 870 larvae assayed, 17 were found to be infected. All the infected larvae appeared to be Simnulium variegatum, the dominant species during the sampling period. IV infections were common in the spring (17–37% depending on site) but appeared absent in the S. variegatum population for most of the summer months, reappearing again in the autumn (0–20% infected). These fluctuations were concurrent with biotic and abiotic factors: elevated levels of covert infection occurred at low population densities, high water flow rates, low temperatures (and presumably slower growth rates), although it is not clear if any cause-and-effect relationship exists. Patent infections occurred immediately after the peak of covert infection in the spring, and again in the autumn. Virus characterization of isolates from covertly infected larvae showed that three distinct groups of isolates were present in the blackfly population. Isolates from the springtime populations were mostly variants of an isolate found in patently infected blackfly larvae in the 1970s (Aberystwyth IV). Isolates from the autumn populations were mostly variants of an isolate from a patently infected larva found in September the previous year. A third group comprised a single novel isolate which was detected in a covertly infected larva. The mechanisms by which IVs persist in blackfly populations remain unknown, although the role of alternative hosts is a possibility which needs to be studied.     

The mode of transmission of Tipula iridescent virus: I. Source of infection

A virus was isolated from a diseased tipulid larva and identified as Tipula iridescent virus (TIV) on the basis of the size and morphology of the virion, the production of iridescence in vitro and in infected tipulid larvae, and a serological reaction between antiserum against the virus and an isolate of TIV.A stock of Tipula oleracea was bred in the laboratory. Subjection of larvae to several stress factors did not result in any evidence for activation of a latent virus. Healthy T. oleracea larvae did not develop iridescence when confined in petri dishes with either live TIV-infected larvae or with large amounts of their feces, although these feces were found to contain infective virus by injecting extracts into healthy larvae. It appears that the concentration of virus in the feces of infected larvae is not high enough for them to serve as a source of infection. It was shown that the cadavers of TIV-infected larvae can serve as a source of infection for healthy first- and fourth-instar larvae.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.     

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C₆₀ fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1?h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID₅₀/mL. The influence of the C₆₀ concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001?mg/mL. It has been found that C₆₀ is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C₆₀ fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.     

The mode of transmission of Tipula iridescent virus: II. Route of infection

Tipula iridescent virus (TIV) was inoculated into Tipula oleracea larvae via different routes. It was found to be much more infective when it was injected into the hemocoel than when it was ingested by the larvae.T. oleracea embryos did not become infected when eggs were laid in agar containing TIV, and there was no evidence that larvae can become infected via the spiracles or that transovum transmission occurs. It is suggested that TIV is transmitted principally by cannibalism, including killing and ingesting infected larvae, and finding dead infected larvae and ingesting them. It is proposed that a new generation becomes infected by first-instar larvae feeding upon infected fourth-instar larvae which have survived from the previous generation.     

Infection of the boll weevil by Chilo iridescent virus

When infections with Chilo iridescent virus (CIV) were induced in larvae of boll weevils, Anthonomus grandis, by intrahemocoelic injection or by feeding, and in adults by feeding, the typical blue coloration of adipose tissue developed at 3–7 days postinfection, and mortality occurred after 3 days. The symptomatology and the pathological expressions depended on the initial infectious titer. The virus remained viable when it was added to the feeding stimulant bait used to infect weevils with protozoan pathogens in the field, and weevils feeding on the formulation became infected when it had been exposed 1–3 days in nature.     

An iridescent virus of the bollworm Heliothis zea (Lepidoptera:Noctuidae)

Larvae of Heliothis zea exhibiting iridescent lavender-blue, blue, blue-green color were obtained in spring field collections in host plants on a road right-of-way in Bolivar County, Mississippi. An iridescent virus was isolated and purified from these larvae. Size range of the virus was 131 to 160 nm, with a mean ± SD of 145 ± 6.5. The nucleic acid content was: DNA, 13.97 ± 1.58% (0.139 μg of DNA/mg of viral protein); RNA, none. Experimental infection was not achieved per os, but the virus was highly virulent by intrahemocoelic injection. The virus from larvae killed in this way was of the same size and morphology as that obtained in the initial isolation.     

Effects of an optical brightener and an abrasive on iridescent virus infection and development of Aedes aegypti

The insecticidal properties of certain entomopathogenic viruses can be greatly improved in mixtures with substances that affect the integrity of the insect peritrophic membrane, particularly optical brighteners. We aimed to determine the effect of an optical brightener, Blankophor BBH, and an abrasive compound, silicon carbide, alone and in mixtures, on the prevalence of patent and covert infection of Aedes aegypti (L.) (Diptera: Culicidae) by Invertebrate iridescent virus 6 (IIV‐6) (Iridoviridae). The prevalence of patent infection by IIV‐6 was < 1.5% in all treatments involving virus. Contrary to predictions, there were significantly fewer patent infections in virus treatments involving Blankophor with or without silicon carbide compared with controls. Covert infection of adults detected by insect bioassay was between 6.7 and 12.2%, although no significant differences were observed between treatments. Exposure to IIV‐6 alone or silicon carbide alone did not significantly increase larval mortality compared to the controls, whereas exposure to Blankophor alone, or in any combination with IIV‐6 or silicon carbide, clearly increased larval mortality. These effects did not carry‐over to the pupal stage. Adult females emerged ～1.5 days later than males. Compared to control insects, female development rate was extended by 11.4 and 12.6% in the treatments involving IIV‐6 alone and silicon carbide alone, respectively. The sex ratio at adult emergence did not differ significantly between control insects and those of other treatments. These results support the hypothesis that the gut is unlikely to represent the principal point of infection of mosquito larvae by iridescent viruses.     

The effect of temperature upon Tipula iridescent virus infection in Tipula oleracea

The mean incubation period (time from inoculation with virus to first appearance of iridescence) was used as an indication of the rate of replication of Tipula iridescent virus (TIV) in Tipula oleracea larvae. The mean incubation period and survival time (time from inoculation with virus to death) were compared with the mean instar duration at a series of temperatures. In most stages of the insect the optimum temperature for the replication of TIV and the temperature for the shortest mean survival time coincided with the peak temperature (the temperature for the fastest development of the insect stage). The peak temperature for T. oleracea does not appear to be the same for each stage, and the optimum temperature for TIV replication appears to be closely linked to the peak temperature of the infected stage. The optimum temperature (the temperature at which most individuals survived from hatching to the adult stage) of the insect was 20°C. Tipula iridescent virus replicated in T. oleracea larvae and pupae at 3° and 27°C, which are near the temperature limits for the insect. Incubation periods and survival times in TIV-inoculated larvae incubated in the field were much longer in winter than in summer.     

Establishment of a loop‐mediated isothermal amplification system for on‐site diagnosis of Oryctes rhinoceros nudivirus in Allomyrina dichotoma (Coleoptera: Scarabaeidae)

The Korean insect industry is rapidly developing, with the Korean horn beetle Allomyrina dichotoma as one of the most popular insects kept as pets. Korean horn beetles reared in local farms are suffering from Oryctes rhinoceros nudivirus (OrNV). In a nationwide investigation on the early death of young larvae due to this virus, 70 to 77% mortality was found. It was also confirmed that several larvae collected from some wild horn beetle habitats were infected with the virus. Thus, it was concluded that the virus could be transmitted vertically from an infected adult to the offspring and that the OrNV disease is a very serious threat to the wild horn beetle in Korea. With local farmers expecting that an early detection technique would be helpful for the quick removal of infected larvae, an on‐site diagnosis method for the viral disease using loop‐mediated isothermal amplification (LAMP) assay was thus tested. To avoid the DNA extraction process, the LAMP assay used a 50‐fold diluted hemolymph was set for diagnosis standardization and convenient on‐site application to infected larvae from local farms. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting the OrNV_gp102 gene. To evaluate the LAMP specificity, two bacterial pathogens, Bacillus thuringiensis and Serratia marcescens, causing disease in A. dichotoma were tested along with OrNV. The positive results were detected only from the OrNV‐infected larvae.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

Transmission of a baculovirus in populations of Oryctes rhinoceros

The transmission of the baculovirus of Oryctes rhinoceros, previously called Rhabdionvirus oryctes, was studied. O. rhinoceros adults became infected with the virus when kept in a mixture of sawdust and ground-up virus-killed larvae or together with other virus-infected adults. In the field, mated females were more frequently infected than unmated females. Adults developing from larvae that had survived exposure to various dosages of the virus were not infected. No virus infections occurred in larvae hatching from eggs surface-contaminated with the virus. Larvae hatching from eggs laid by virus-infected females very rarely were infected.In the O. rhinoceros population the virus is transmitted most frequently during mating, possibly when the uninfected partner contacts virus material excreted by the infected partner. The virus can be transmitted in a similar way when infected and healthy beetles feed together in palm trees. Beetles visiting larval breeding sites containing freshly virus-killed larvae can become infected. Virus-infected beetles can pass the infection to healthy larvae when visiting a breeding site.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

Incidence and pathology of common viruses of nerine in Tasmania

This is the first study of the incidence of common viruses within bulb stocks of nerine in Australia. Five viruses were frequently found within commercial stocks but were never associated with obvious viral symptom expression in the field. This study found cucumber mosaic virus (CMV) infections of Nerine flexuosa, and nerine virus Y (NeVY) infections of N. flexuosa, N. bowdenii and N. fothergilli which we believe to be new host records. Virus incidence varied with virus species, host species, and sample site but did not vary within the same bulb stocks over a 2 yr period. Incidence of the major viruses appeared to correlate with the efficiency of transfer from mother bulb to offsets, suggesting that virus status in Tasmania may be largely determined by the incidence of viruses within original planting material and by subsequent vegetative spread. No effect of the most frequent virus infections was found on the efficiency of flowering, flower quality, and dormant bulb fresh weight in N. flexuosa and N. fothergilli with the exception of a small (16.8%), but economically unimportant decrease in scape length of N. flexuosa co‐infected with NeLV and NeVY. It is concluded that current virus infections in nerine in Tasmania pose little commercial significance to the developing cut flower industry, however discovery of NeYSV in one sample of Lycoris aurea warrants monitoring to ensure that spread of this important pathogen does not occur.