Analysis of the Role of Bradysia impatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana.

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

Tropism, pathology, and transmission of equine parvovirus-hepatitis.

Equine parvovirus-hepatitis (EqPV-H) has recently been associated with cases of Theiler's disease, a form of fulminant hepatic necrosis in horses. To assess whether EqPV-H is the cause of Theiler's disease, we first demonstrated hepatotropism by PCR on tissues from acutely infected horses. We then experimentally inoculated horses with EqPV-H and 8 of 10 horses developed hepatitis. One horse showed clinical signs of liver failure. The onset of hepatitis was temporally associated with seroconversion and a decline in viremia. Liver histology and in situ hybridization showed lymphocytic infiltrates and necrotic EqPV-H-infected hepatocytes. We next investigated potential modes of transmission. Iatrogenic transmission via allogeneic stem cell therapy for orthopedic injuries was previously suggested in a case series of Theiler's disease, and was demonstrated here for the first time. Vertical transmission and mechanical vectoring by horse fly bites could not be demonstrated in this study, potentially due to limited sample size. We found EqPV-H shedding in oral and nasal secretions, and in feces. Importantly, we could demonstrate EqPV-H transmission via oral inoculation with viremic serum. Together, our findings provide additional information that EqPV-H is the likely cause of Theiler's disease and that transmission of EqPV-H occurs via both iatrogenic and natural routes.

Assessing the feasibility of fly based surveillance of wildlife infectious diseases.

Monitoring wildlife infectious agents requires acquiring samples suitable for analyses, which is often logistically demanding. A possible alternative to invasive or non-invasive sampling of wild-living vertebrates is the use of vertebrate material contained in invertebrates feeding on them, their feces, or their remains. Carrion flies have been shown to contain vertebrate DNA; here we investigate whether they might also be suitable for wildlife pathogen detection. We collected 498 flies in Taï National Park, Côte d'Ivoire, a tropical rainforest and examined them for adenoviruses (family Adenoviridae), whose DNA is frequently shed in feces of local mammals. Adenoviral DNA was detected in 6/142 mammal-positive flies. Phylogenetic analyses revealed that five of these sequences were closely related to sequences obtained from local non-human primates, while the sixth sequence was closely related to a murine adenovirus. Next-generation sequencing-based DNA-profiling of the meals of the respective flies identified putative hosts that were a good fit to those suggested by adenoviral sequence affinities. We conclude that, while characterizing the genetic diversity of wildlife infectious agents through fly-based monitoring may not be cost-efficient, this method could probably be used to detect the genetic material of wildlife infectious agents causing wildlife mass mortality in pristine areas.

First isolation of a Marseillevirus in the Diptera Syrphidae Eristalis tenax.

OBJECTIVE: Giant viruses and amoebae are common in freshwater, where they can coexist with various insects. We screened insect larvae to detect giant viruses using a high-throughput method. METHODS: We analyzed 86 Eristalis tenax larvae obtained from stagnant water reservoirs in Tunisia. The larvae were decontaminated and then dissected to remove internal parts for coculture with Acanthamoeba polyphaga. Genome sequencing of isolated viruses was performed on a 454 Roche instrument, and comparative genomics were performed. RESULTS: One Marseillevirus, named Insectomime virus, was isolated. The genome assembly generated two scaffolds, which were 382,776 and 3,855 bp in length. Among the 477 identified predicted proteins, the best hit for 435 of the identified proteins was a Marseillevirus or Lausannevirus protein. Tunisvirus was the most closely related to Insectomime, with 446 orthologs. One Insectomime protein shared with Lausannevirus and Tunisvirus showed the highest similarity with a protein from an aphid. CONCLUSION: The isolation of a Marseillevirus from an insect expands the diversity of environments in which giant viruses have been isolated. The coexistence of larvae and giant viruses in stagnant water may explain the presence of the giant virus in the larva internal structures. This study illustrates the putative role of amoeba in lateral gene transfer not only between the organisms it phagocytoses, but also between organisms living in the same environment. © 2013 S. Karger AG, Basel.

Distribution of 'promoter' sandflies associated with incidence of classic Kaposi's sarcoma.

The patchy geographical distributions of classic Kaposi's sarcoma (KS) and human herpesvirus type 8 (HHV-8), better known as Kaposi's sarcoma-associated herpesvirus (KSHV) remain unexplained. It has been proposed that certain species of bloodsucking insects ('promoter arthropods') promote the reactivation of HHV-8/KSHV and facilitate both HHV-8/KSHV transmission and KS development. This hypothesis was tested by sampling the presence and density of human-biting Diptera with CDC light traps in two areas of Sardinia with contrasting incidence rates of classic KS. In total, 11,030 specimens (99.9% sandflies and 0.1% mosquitoes) belonging to 10 species were collected from 40 rural sites. Five of these species are considered to be possible promoter arthropods because of the irritation their bites cause: Phlebotomus perniciosus Newstead; Phlebotomus perfiliewi Parrot (Diptera: Psychodidae); Aedes berlandi Seguy; Culiseta annulata (Schrank) and Culex theileri Theobald (Diptera: Culicidae). Five species are probable 'non-promoters' because their bites are not particularly irritating: Culiseta longiareolata (Macquart); Culex pipiens s.l.; Anopheles algeriensis Theobald; Anopheles maculipennis s.l., and Anopheles plumbeus Stephens. A significant correlation was found between the geographical distribution of promoter arthropods and incidence rates of KS (Spearman's r = 0.59,P < 0.01). Promoter arthropods were more likely to be caught in areas with cutaneous leishmaniasis and a past high prevalence of malaria, and in areas of limestone, acid volcanic soil and cereal cultivation. The study supports the association between promoter arthropods and classic KS, which may explain the geographic variability of KS and HHV-8/KSHV, and highlights the links with a number of variables previously associated with the incidence of KS.

Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice.

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.

The detection of Bovine Papillomavirus type 1 DNA in flies.

BPVs are double stranded DNA viruses that can infect several species other than the natural host, cattle, including equids. In equids, BPV-1, and, less commonly BPV-2, infection gives rise to fibroblastic tumours of the skin. Whilst a causal relationship between BPV-1/2 and equine sarcoids is now well established, how the disease is transmitted is not known. In this study we show BPV-1 DNA can be detected in flies trapped in the proximity of sarcoid-affected animals. Sequence analysis of the BPV-1 LCR from flies indicates that flies harbour BPV-1 LCR sequence variants II and IV which are commonly detected in equine sarcoids. These data suggest that flies may be able to transmit BPV-1 between equids.

Detection and isolation of exotic Newcastle disease virus from field-collected flies.

Flies were collected by sweep net from the vicinity of two small groups of "backyard" poultry (10-20 chickens per group) that had been identified as infected with exotic Newcastle disease virus (family Paramyxoviridae, genus avulavirus, ENDV) in Los Angeles County, CA, during the 2002-2003 END outbreak. Collected flies were subdivided into pools and homogenized in brain-heart infusion broth with antibiotics. The separated supernatant was tested for the presence of ENDV by inoculation into embryonated chicken eggs. Exotic Newcastle disease virus was isolated from pools of Phaenicia cuprina (Wiedemann), Fannia canicularis (L.), and Musca domestica L., and it was identified by hemagglutination inhibition with Newcastle disease virus antiserum. Viral concentration in positive pools was low (<1 egg infectious dose50 per fly). Isolated virus demonstrated identical monoclonal antibody binding profiles as well as 99% sequence homology in the 635-bp fusion gene sequence compared with ENDV recovered from infected commercial egg layer poultry during the 2002 outbreak.

A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae).

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.

Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex.

Tomato yellow leaf curl disease (TYLCD) is caused by a group of geminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complex and are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causes great yield losses in many countries throughout the Mediterranean region and the Middle East. In this study, the efficacy of post-transcriptional gene silencing (PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curl virus-mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curl Malaga virus, and tomato yellow leaf curl Sardinia virus-Spain [2] were selected and used to design a construct that can trigger broad resistance against different viruses that cause tomato yellow leaf curl disease. The silencing construct was cloned into an Agrobacterium-binary vector in sense and antisense orientation and used in transient assay to infiltrate tomato and Nicotiana benthamiana plants. A high level of resistance was obtained when plants were agro-infiltrated with an infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or challenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16-20 days post infiltration with the silencing construct. Results of the polymerase chain reaction showed that the resistance was effective against all three viruses. Furthermore, dot blot hybridization and PCR failed to detect viral DNA in symptomless, silenced plants. A positive correlation between resistance and the accumulation of TYLCV-specific siRNAs was observed in silenced plants. Together, these data provide compelling evidence that PTGS can be used to engineer geminivirus-resistant plants.

Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure.

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.

Fossil evidence of insect pathogens.

The present report describes fossil evidence of insect pathogens, heretofore, almost non-existent, from six samples of amber ranging in age from 15 to 100 million years. They include a cytoplasmic polyhedrosis virus and trypanosomatid infection in an adult biting midge (Diptera: Ceratopogonidae), and a nuclear polyhedrosis virus in an adult sand fly (Diptera: Phlebotomidae), both from Early Cretaceous Burmese amber, several types of fungal thalli on the cuticle of an adult mosquito (Culicidae: Diptera), as well as a fungal growth on the prothorax of a fungus gnat (Mycetophilidae: Diptera) in Dominican amber and large tumors in the body cavity of a caterpillar (Lepidoptera) in Mexican amber. These discoveries suggest that insect polyhedrosis viruses were present 100 million years ago and present the possibility that vertebrate arboviruses (especially those in the family Reoviridae) could have evolved from cytoplasmic polyhedrosis viruses infecting biting insects. The flagellates in the Early Cretaceous biting midge represent the first fossil record of monogenetic trypanosomatid infections of arthropods.

Relatedness of baculovirus and gypsy retrotransposon envelope proteins.

BACKGROUND: Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins. One group utilizes gp64, a low pH-dependent envelope fusion protein, whereas the other employs a protein family (e.g. LD130 in the Lymantria dispar nucleopolyhedrovirus) unrelated to gp64, but that is also low pH-dependent. Database searches with members of the LD130 protein family often record significant levels of homology to envelope proteins from a number of insect retrovirus-like transposable elements of the gypsy class. In this report, the significance of the homology between these two types of envelope proteins is analyzed. RESULTS: The significance of the alignment scores was evaluated using Z-scores that were calculated by comparing the observed alignment score to the distribution of scores obtained for alignments after one of the sequences was subjected to 100 random shuffles of its sequence. These analyses resulted in Z-scores of >9 for members of the LD130 family when compared to most gypsy envelope proteins. Furthermore, in addition to significant levels of sequence homology and the presence of predicted signal sequences and transmembrane domains, members of this family contain a possible a furin cleavage motif, a conserved motif downstream of this site, predicted coiled-coil domains, and a pattern of conserved cysteine residues. CONCLUSIONS: These analyses provide a link between envelope proteins from a group of insect retrovirus-like elements and a baculovirus protein family that includes low-pH-dependent envelope fusion proteins. The ability of gypsy retroelements to transpose from insect into baculovirus genomes suggests a pathway for the exchange of this protein between these viral families.

Laboratory vector competence of black flies (Diptera:Simuliidae) for the Indiana serotype of vesicular stomatitis virus.

In previous experiments we have demonstrated that colonized and wild black flies are competent laboratory vectors of different Mexican and Western USA isolates of vesicular stomatitis virus, serotype New Jersey (VSV-NJ). We have recently demonstrated biological VSV-NJ transmission by black flies using animal models. In the study described here, we tested the vector competence of colonized and wild black flies for the vesicular stomatitis virus, serotype Indiana (VSV-IN). A 1998 equine isolate was used. After a 10 day incubation period, saliva from experimentally infected Simulium vittatum and S. notatum was individually collected and tested for the presence of infectious virus. Virus was detected in the saliva of both species following oral infection, indicating that they are competent laboratory vectors of VSV-IN. In addition, the results suggest that the black fly gut may exert evolutionary pressures on the virus.

Amino acids in the capsid protein of tomato yellow leaf curl virus that are crucial for systemic infection, particle formation, and insect transmission.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

Pantropic retroviral vectors mediate somatic cell transformation and expression of foreign genes in dipteran insects.

The control of insects that transmit disease and damage crops has become increasingly difficult. The ability to genetically engineer insects would facilitate strategies to protect crops and block arthropod vector-borne disease transmission. Transformation vectors based on insect transposable elements have been developed, but most have limited host ranges. A promising alternative is the pantropic retroviral vector, which is packaged with the envelope glycoprotein from vesicular stomatitis virus and is replication-defective. We show here that pantropic murine retroviral vectors can mediate high-level expression of foreign genes in somatically transformed insect larvae and adults of three dipteran genera. This success demonstrates the potential for germline transformation mediated by pantropic retroviral vectors.

Characterization of iridovirus IV1 polypeptides: mapping by surface labelling.

A comprehensive index of IV1(Tipula iridescent virus, or TIV)-associated polypeptides has been established using enrichments of "empty" and "filled" virions that are considered to be maturation-phase-related. The mapping strategy which involved one- and two-dimensional polyacrylamide gel electrophoresis, silver staining, disulphide bond reduction and 125I iodination of putative surface proteins revealed 103 and 116 polypeptides for empty and filled capsids, respectively. These estimates could be reduced to < or = 70 by reclassing multicharged polypeptides of approximately identical masses as single entitles. At least 10 polypeptides from empty and filled virions were involved in intermolecular sulphhydryl linkages and another 11 species were identified as putative outer shell (surface) polypeptides. These data provide useful criteria for iridovirus classification and identification of candidate polypeptides involved in capsid formation and maturation.

Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA.

One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny.

Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed.

A full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.