Rice yellow stunt virus activates polyamine biosynthesis to promote viral propagation in insect vectors by disrupting ornithine decarboxylase antienzyme function.

Intracellular polyamines (putrescine, spermidine, and spermine) have emerged as important molecules for viral infection; however, how viruses activate polyamines biosynthesis to promote viral infection remains unclear. Ornithine decarboxylase 1 (ODC1) and its antienzyme 1 (OAZ1) are major regulators of polyamine biosynthesis in animal cells. Here, we report that rice yellow stunt virus (RYSV), a plant rhabdovirus, could activate putrescine biosynthesis in leafhoppers to promote viral propagation by inhibiting OAZ1 expression. We observed that the reduction of putrescine biosynthesis by treatment with difluormethylornithine (DFMO), a specific nontoxic inhibitor of ODC1, or with in vitro synthesized dsRNAs targeting ODC1 mRNA could inhibit viral infection. In contrast, the supplement of putrescine or the increase of putrescine biosynthesis by treatment with dsRNAs targeting OAZ1 mRNA could facilitate viral infection. We further determined that both RYSV matrix protein M and ODC1 directly bind to the ODC-binding domain at the C-terminus of OAZ1. Thus, viral propagation in leafhoppers would decrease the ability of OAZ1 to target and mediate the degradation of ODC1, which finally activates putrescine production to benefit viral propagation. This work reveals that polyamine-metabolizing enzymes are directly exploited by a vector-borne virus to increase polyamine production, thereby facilitating viral infection in insect vectors.

New insights on the transmission mechanism of tenuiviruses by their vector insects.

Tenuiviruses, which cause serious diseases in rice, wheat, maize and other gramineae crops, recently have been assigned to the family Phenuiviridae in the order Bunyavirales. Transmission of tenuiviruses to host plants depends on the specific vector planthoppers. The interaction between the virus and insect offers critical points for developing an efficient management strategy. This review focuses on recent advancements in our understanding of the interactions between the virus and insect components. Vector components such as various proteins play major roles in virus replication, stability and transovarial transmission. The virus can either directly interact with these proteins or regulate expression of genes that encode them to alter the metabolism or defense mechanisms of the insect vectors. However, the vector components that are involved in virus infection and movement in midgut and salivary glands are not as well explored and are targets for further study.

Development and use of three monoclonal antibodies for the detection of rice black-streaked dwarf virus in field plants and planthopper vectors.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries. The use of serological methods for RBSDV detection depends on the availability of antibodies. In this study, three highly sensitive and specific murine monoclonal antibodies (MAbs) against RBSDV antigens were produced using crude extracts from tumors of RBSDV-infected maize as the immunogen, and two serological assays, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were developed for RBSDV detection. RESULTS: All three MAbs reacted strongly and specifically with the crude extracts from RBSDV-infected plant and planthopper tissues. The detection endpoints of three MAbs (12E10, 18F10 and 5G5) in ACP-ELISA were respectively 1:40,960, 1:40,960, 1:81,920 (w/v, g mL-1) with the crude extract of infected maize, 1:10,240, 1:20,480, 1:20,480 (w/v, g mL-1) with the crude extract of infected rice, 1:5,120, 1:10,240, 1:10,240 (w/v, g mL-1) with the crude extract of infected wheat, 1:9,600, 1:9,600, 19,200 (individual planthopper/µL) with the crude extract of infected planthopper. The newly developed ACP-ELISA could detect the virus in the infected maize, wheat, rice tissue crude extracts diluted at 1:81,920, 1:20,480, 1:10,240 (w/v, g mL-1), respectively, and in individual viruliferous planthopper extract diluted at 1:19200 (individual planthopper/µL). The dot-ELISA was proved to detect the virus in the infected maize, wheat and rice tissue crude extracts diluted at 1:320 (w/v, g mL-1), and in individual viruliferous planthopper extract diluted at 1:1,600 (individual planthopper/µL), respectively. Field plants (915) and planthopper samples (594) from five provinces of China were screened for the presence of RBSDV using the two developed serological assays. The results indicated that 338 of the 915 plant samples and 19 of the 594 planthopper samples were infected by RBSDV. CONCLUSIONS: The newly developed ACP-ELISA and dot-ELISA were highly sensitive and specific to detect RBSDV in field plant and planthopper samples. The field survey demonstrated that RBSDV is widespread in rice, maize and wheat crops in Jiangsu, Zhejiang, Shandong provinces of China.

Infection of Melanoplus sanguinipes grasshoppers following ingestion of rangeland plant species harboring vesicular stomatitis virus.

Knowledge of the many mechanisms of vesicular stomatitis virus (VSV) transmission is critical for understanding of the epidemiology of sporadic disease outbreaks in the western United States. Migratory grasshoppers [Melanoplus sanguinipes (Fabricius)] have been implicated as reservoirs and mechanical vectors of VSV. The grasshopper-cattle-grasshopper transmission cycle is based on the assumptions that (i) virus shed from clinically infected animals would contaminate pasture plants and remain infectious on plant surfaces and (ii) grasshoppers would become infected by eating the virus-contaminated plants. Our objectives were to determine the stability of VSV on common plant species of U.S. Northern Plains rangelands and to assess the potential of these plant species as a source of virus for grasshoppers. Fourteen plant species were exposed to VSV and assayed for infectious virus over time (0 to 24 h). The frequency of viable virus recovery at 24 h postexposure was as high as 73%. The two most common plant species in Northern Plains rangelands (western wheatgrass [Pascopyrum smithii] and needle and thread [Hesperostipa comata]) were fed to groups of grasshoppers. At 3 weeks postfeeding, the grasshopper infection rate was 44 to 50%. Exposure of VSV to a commonly used grasshopper pesticide resulted in complete viral inactivation. This is the first report demonstrating the stability of VSV on rangeland plant surfaces, and it suggests that a significant window of opportunity exists for grasshoppers to ingest VSV from contaminated plants. The use of grasshopper pesticides on pastures would decrease the incidence of a virus-amplifying mechanical vector and might also decontaminate pastures, thereby decreasing the inter- and intraherd spread of VSV.

Grasshoppers (Orthoptera: Acrididae) could serve as reservoirs and vectors of vesicular stomatitis virus.

Vesicular stomatitis (VS) is an economically devastating disease of livestock in the Americas. Despite strong circumstantial evidence for the role of arthropods in epizootics, no hematophagous vector explains the field evidence. Based on the spatiotemporal association of grasshopper outbreaks and VS epizootics, we investigated the potential role of these insects as vectors and reservoirs of the disease. The critical steps in the grasshopper-bovine transmission cycle were demonstrated, including 1) 62% of grasshoppers [Melanoplus sanguinipes (F.)] fed vesicular stomatitis virus (VSV) from cell culture became infected, with titers reaching 40,000 times the inoculative dose; 2) 40% of grasshoppers that cannibalized VSV-infected grasshopper cadavers became infected, amplifying virus up to 1,000-fold; 3) one of three cattle consuming VSV-infected grasshopper cadavers contracted typical VS and shed virus in saliva; and 4) 15% of grasshoppers became infected when fed saliva from this infected cow. The ecological conditions and biological processes necessary for these transmissions to occur are present throughout much of the Americas. Field studies will be required to show these findings are relevant to the natural epidemiology of VSV.

The spheroidin of an entomopoxvirus isolated from the grasshopper Anacridium aegyptium (AaEPV) shares low homology with spheroidins from lepidopteran or coleopteran EPVs.

Based on virion morphology, the current virus taxonomy groups entomopoxviruses (EPVs) (Poxvirus: Entomopoxvirinae) from coleopteran and dipteran hosts in separated genera, wilts it keeps viruses infecting either lepidopteran or orthopteran hosts in the same genus. In contrast to the morphological criteria, the few data available from recent studies at the genetic level have suggested that EPVs infecting different insect orders are phylogenetically distant. In order to elucidate EPVs phylogeny we have cloned and sequence the highly conserved/highly expressed spheroidin gene of Anacridium aegyptium entomopoxvirus (AaEPV). This gene and its promoter is of interest for the development of genetic engineering on EPVs. The spheroidin gene was located in the AaEPV genome by Southern blot and hybridisation with specific degenerated oligonucleotides probes synthesised after partial sequencing of the purified spheroidin protein. A total of 3489 bp were sequenced. This sequence included the coding and promoter region of 969 residues 108. 8 kDa protein identified as spheroidin. AaEPV spheroidin contains 21 cysteine residues (2.2%) and 14 N-glycosylation putative sites distributed along the sequence. The cysteine residues are particularly abundant at the C-terminal end of the protein, with 11 residues in the last 118 aa. Our results confirm that the spheroidin is highly conserved only between EPVs isolated from the same insect order. Polyclonal antibodies raised against AaEPV spherules specifically revealed spheroidin in Western Blots failing to cross-react with MmEPV or AmEPV spheroidins or MmEPV fusolin. Comparison of spheroidins at the aa level demonstrate that AaEPV spheroidin shares only 22.2 and 21.9% identity with the lepidopteran AmEPV and the coleopteran MmEPV spheroidins, respectively, but 82.8% identity with the orthopteran MsEPV spheroidin. Only two highly conserved domains containing the sequence (V/Y)NADTG(C/L) and LFAR(I/A) have been identified in all known spheroidins. The phylogenetic tree constructed according to the CLUSTALX analysis program revealed that EPVs are clearly separated in three groups - lepidopteran, coleopteran and orthopteran - according to the insect order of the virus hosts. In base to our results, the split of the genus Entomopoxvirus B dissociating lepidopteran and orthopteran EPVs into two different genera is suggested.

Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates.

Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 0.25. The introduction of a 5'-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to >/=30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a approximately 10(-4) decrement in the rate of topoisomerase-catalyzed attack of 5'-SH DNA on the DNA-(3'-phosphotyrosyl)-enzyme intermediate. 5'-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on k(cl).

Spodoptera littoralis type B nucleopolyhedrovirus infection of a grasshopper cell line.

We determined that the type B nucleopolyhedrovirus of the Egyptian cottonworm, Spodoptera littoralis (SpliNPV), can infect a cell line derived from a grasshopper. We compared the infectivity of SpliNPV in two lepidopteran cell lines (Sf9 and Md210) and in a cell line (MSE4) derived from the western migratory grasshopper, Melanoplus sanguinipes (Orthoptera: Acrididae). Both Sf9 and MSE4 cells were permissive for SpliNPV replication and supported production of viable progeny. Md210 cells were nonpermissive for SpliNPV, and although the virus entered into these cells, they supported neither viral replication nor production of viable progeny. Infection of MSE4 cells with SpliNPV resulted in cytopathic effects within 48 h post infection and complete destruction of the cells within 5 days. Both virions and polyhedra were detected within virus-infected MSE4 cells by transmission electron microscopy. Extracellular virions were detected in the culture medium and were infectious to Sf9 cells, indicating that the MSE4 cells supported production of viable virus progeny.

The genome of Melanoplus sanguinipes entomopoxvirus.

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.

A polymerase chain reaction for the detection of nucleopolyhedroviruses in infected insects: the fate of the Spodoptera littoralis virus in Locusta migratoria.

The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) is a potential pest control agent of Spodoptera spp. As part of our studies to establish the use of this virus, a polymerase chain reaction (PCR)-based method was developed for the detection of viral DNA in infected insects. PCR amplification of the polyhedrin sequences enabled the detection of low levels of viral DNA directly from viral occlusion bodies or from total larval DNA. The use of different sets of synthetic DNA primers allowed us to differentiate between SlNPV and the Autographa californica NPV (AcNPV) and to identify a new AcNPV variant isolated from a cotton pest, Pectinophora gossypiella NPV. The PCR method was also used to test for the possible infection of Locusta migratoria larvae by SlNPV, reported by Bensimon et al., 1987. The progress of SlNPV infection in L. migratoria larvae was monitored by PCR for 2 weeks. The reaction revealed decreasing amounts of viral DNA in infected larvae. During this time, no signs of disease were observed in the infected locusts.