Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict.

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Baculovirus ODV-E66 degrades larval peritrophic membrane to facilitate baculovirus oral infection.

ODV-E66 is a major envelope proteins of baculovirus occlusion derived virus (ODV) with chondroitinase activity. Here, we studied the roles of ODV-E66 during Helicoverpa armigera nucleopolyhedrovirus (HearNPV) primary infection. ODV-E66 is a late viral protein dispensable for BV production and ODV morphogenesis. Deletion of odv-e66 had a profound effect on HearNPV oral infectivity in 4th instar larvae with a 50% lethal concentration (LC50) value of 26 fold higher than that of the repaired virus, compared to in 3rd instar larvae. Calcofluor white, an agent which destroys the peritrophic membrane (PM), could rescue the oral infectivity of odv-e66 deleted HearNPV, implying the PM may be the target of ODV-E66. In vitro assays showed HearNPV ODV-E66 has chondroitinase activity. Electron microscopy demonstrated that odv-e66 deletion alleviated the damage to the PM caused by HearNPV infection. These data suggest an important role of ODV-E66 in the penetration of the PM during oral infection.

3H-117, a structural protein of Heliothis virescens ascovirus 3h (HvAV-3h).

The open reading frame 117 (3h-117) of Heliothis virescens ascovirus 3h (HvAV-3h), which is a conserved coding region present in all completely sequenced ascovirus members, was characterized in this study. By RT-PCR detection, 3h-117 transcription began at 6-h post-infection (hpi) and remained stable until 168 hpi in HvAV-3h-infected Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. In addition, 3h-117 putatively encodes a 21.5-kDa protein (3H-117) predicted to be a CTD-like phosphatase. Western blot analysis using a prepared rabbit polyclonal antibody specific to 3H-117 showed that the product could be detected at 24 hpi, which remained stably detectable until 168 hpi. The same analysis also demonstrated that the 3H-117 protein localized in the virions of HvAV-3h. Immunofluorescence analysis showed that at 24 hpi, 3H-117 was mainly located in the nuclei of H. armigera larval fat body cells and later spread into the cytoplasm. In summary, our results indicate that 3H-117 is a structural protein of HvAV-3h.

Providence virus: An animal virus that replicates in plants or a plant virus that infects and replicates in animal cells?

INTRODUCTION: Emerging viral diseases, most of which are zoonotic, pose a significant threat to global health. There is a critical need to identify potential new viral pathogens and the challenge is to identify the reservoirs from which these viruses might emerge. Deep sequencing of invertebrate transcriptomes has revealed a plethora of viruses, many of which represent novel lineages representing both plant and animal viruses and little is known about the potential threat that these viruses pose. METHODS: Providence virus, an insect virus, was used to establish a productive infection in Vigna unguiculata (cowpea) plants. Providence virus particles purified from these cowpea plants were used to infect two mammalian cell lines. FINDINGS: Here, we present evidence that Providence virus, a non-enveloped insect RNA virus, isolated from a lepidopteran midgut cell line can establish a productive infection in plants as well as in animal cells. The observation that Providence virus can readily infect both plants and mammalian cell culture lines demonstrates the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants and invertebrate and vertebrate animal cell lines. CONCLUSIONS: The study highlights the potential of phytophagous insects as reservoirs for viral re-assortment and that plants should be considered as reservoirs for emerging viruses that may be potentially pathogenic to humans.

Genome Characteristics of the Cyclophragma Undans Nucleopolyhedrovirus: A Distinct Species in Group I of Alphabaculovirus.

The Cyclophragma undans nucleopolyhedrovirus (CyunNPV), a potential pest control agent, was isolated from Cyclophragma undans (Lepidoptera: Lasiocampidae), an important forest pest. In the present study, we performed detailed genome analysis of CyunNPV and compared its genome to those of other Group I alphabaculoviruses. Sequencing of the CyunNPV genome using the Roche 454 sequencing system generated 142,900 bp with a G + C content of 45%. Genome analysis predicted a total of 147 hypothetical open reading frames comprising 38 baculoviral core genes, 24 lepidopteran baculovirus conserved genes, nine Group I Alphabaculovirus conserved genes, 71 common genes, and five genes that are unique to CyunNPV. In addition, the genome contains 13 homologous repeated sequences (hrs). Phylogenetic analysis groups CyunNPV under a distinct branch within clade "a" of Group I in the genus Alphabaculovirus. Unlike other members of Group I, CyunNPV harbors only nine of the 11 genes previously determined to be specific to Group I viruses. Furthermore, the CyunNPV lacks the tyrosine phosphatase gene and the ac30 gene. The CyunNPV F-like protein contains two insertions of continuous polar amino acids, one at the conventional fusion peptide and a second insertion at the pre-transmembrane domain. The insertions are likely to affect the fusion function and suggest an evolutionary process that led to inactivation of the F-like protein. The above findings imply that CyunNPV is a distinct species under Group I Alphabaculovirus.

HearNPV Pseudotyped with PIF1, 2, and 3 from MabrNPV: Infectivity and Complex Stability.

Effective oral infection is set off by interaction of a group of conserved per os infectivity factors (PIFs) with larval midgut columnar epithelial cells. We constructed pseudotyped viruses by substituting pif1, pif2 or pif3 genes of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) with their homologs from Mamestra bracissae multiple nucleopolyhedrovirus and tested their infectivity to tissue culture cells and to larvae. Transfection and infection assays revealed that all recombinant viruses generated infectious budded virus in both cell culture and in larvae. Electron microscopy showed synthesized occlusion body and occlusion derived virus (ODV) were morphologically indistinguishable from those of the parental virus. By contrast, feeding assays revealed that pseudotyped viruses could not rescue oral infectivity except for pif3 pseudotyped virus that only partially rescued oral infectivity but at a mortality rate much lower than that of the parental HearNPV. Consistent with the bioassay result, PIF complex was detected in ODVs of pif3 pseudotyped virus only but not in pif1 or pif2 pseudotyped viruses. Our results suggest that PIF complex is essential for oral infectivity, and in the formation of the PIF complex, PIF1, 2 are virus-specific while PIF3 does not appear to be as specific and can function in heterologous environment, albeit to a much more limited extent.

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway.

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

The Complete Genome Sequence of a Second Distinct Betabaculovirus from the True Armyworm, Mythimna unipuncta.

The betabaculovirus originally called Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the characteristic size range and morphology of betabaculovirus granules. Barcoding PCR using cytochrome oxidase I primers with DNA from the MyspGV#8 collection sample confirmed that it had been isolated from the true armyworm, Mythimna unipuncta (Lepidoptera: Noctuidae) and therefore was renamed MyunGV#8. The MyunGV#8 genome was found to be 144,673 bp in size with a nucleotide distribution of 49.9% G+C, which was significantly smaller and more GC-rich than the genome of Pseudaletia unipuncta granulovirus H (PsunGV-H), another M. unipuncta betabaculovirus. A phylogeny based on concatenated baculovirus core gene amino acid sequence alignments placed MyunGV#8 in clade a of genus Betabaculovirus. Kimura-2-parameter nucleotide distances suggested that MyunGV#8 represents a virus species different and distinct from other species of Betabaculovirus. Among the 153 ORFs annotated in the MyunGV#8 genome, four ORFs appeared to have been obtained from or donated to the alphabaculovirus lineage represented by Leucania separata nucleopolyhedrovirus AH1 (LeseNPV-AH1) during co-infection of Mythimna sp. larvae. A set of 33 ORFs was identified that appears only in other clade a betabaculovirus isolates. This clade a-specific set includes an ORF that encodes a polypeptide sequence containing a CIDE_N domain, which is found in caspase-activated DNAse/DNA fragmentation factor (CAD/DFF) proteins. CAD/DFF proteins are involved in digesting DNA during apoptosis.

Midgut-based resistance to oral infection by a nucleopolyhedrovirus in the laboratory-selected strain of the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.

Characterization of two monoclonal antibodies, 38F10 and 44D11, against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus.

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor (F0) and then cleaved into a disulfide-linked F1 and F2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies (mAbs) against the F2 subunit of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) (HaF) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10, recognizes amino acid (aa) 85 to 123 of F2 and the other kind, represented by 44D11, recognizes aa 148 to 173 of F2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the HaF protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF2 that may be involved in the F-mediated membrane fusion process.

Silencing of Mythimna separata chitinase genes via oral delivery of in planta-expressed RNAi effectors from a recombinant plant virus.

OBJECTIVES: To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression. RESULTS: Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %. CONCLUSION: Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.

Mutational and functional analysis of N-linked glycosylation of envelope fusion protein F of Helicoverpa armigera nucleopolyhedrovirus.

The envelope fusion (F) protein of baculoviruses is a heavily N-glycosylated protein that plays a significant role in the virus infection cycle. N-Linked glycosylation of virus envelope glycoprotein is important for virus envelope glycoprotein folding and its function in general. There are six predicted N-glycosylation sites in the F (HaF) protein of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The N-glycosylation site located in the F(2) subunit (N104) of HaF has been identified and functionally characterized previously (Long et al., 2007). In this study, the other five potential N-glycosylation sites located in the HaF1 subunit, namely, N293, N361, N526, N571 and N595, were analysed extensively to examine their N-glycosylation and relative importance to the function of HaF. The results showed that four of these five potential glycosylation sites in the F(1) subunit, N293, N361, N526 and N571, were N-glycosylated in F proteins of mature HearNPV budded viruses (BVs) but that N595 was not. In general, the conserved site N526 was critical to the functioning of HaF, as absence of N-glycosylation of N526 reduced the efficiency of HaF folding and trafficking, consequently decreased fusogenicity and modified the subcellular localization of HaF proteins, and thus impaired virus production and infectivity. The absence of N-glycosylation at other individual sites was found to have different effects on the fusogenicity and subcelluar distribution of HaF proteins in HzAM1 cells. In summary, N-glycosylation plays comprehensive roles in HaF function and virus infectivity, which is further discussed.

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

Complete genome sequence and structural characterization of a novel iflavirus isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae).

Opsiphanes invirae (Lepidopera: Nymphalidae) is a common pest of the African oil palm tree (Elaeis guineensis) in Brazil. Dead larvae were collected in canopy of oil palm trees cultivated in the amazon region (Para State) and analyzed for viral infection. Electron microscopy of caterpillar extracts showed an icosahedral picorna-like virus particle with 30nm in diameter. Total RNA extracted from partially purified virus particles was sequenced. A contig of 10,083 nucleotides (nt) was identified and showed to encode one single predicted polyprotein with 3185 amino acid residues. Phylogenetic analysis showed that the new virus was closely related to another lepidopteran infective virus Spodoptera exigua iflavirus 1(SeIV-1), with 35% amino acid pairwise identity. The novel virus fulfils all ICTV requirements for a new iflavirus species and was named Opsiphanes invirae Iflavirus 1 (OilV-1).

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses.

BACKGROUND: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. RESULTS: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX - Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. CONCLUSIONS: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.

Genome sequence of Erinnyis ello granulovirus (ErelGV), a natural cassava hornworm pesticide and the first sequenced sphingid-infecting betabaculovirus.

BACKGROUND: Cassava (Manihot esculenta) is the basic source for dietary energy of 500 million people in the world. In Brazil, Erinnyis ello ello (Lepidoptera: Sphingidae) is a major pest of cassava crops and a bottleneck for its production. In the 1980s, a naturally occurring baculovirus was isolated from E. ello larva and successfully applied as a bio-pesticide in the field. Here, we described the structure, the complete genome sequence, and the phylogenetic relationships of the first sphingid-infecting betabaculovirus. RESULTS: The baculovirus isolated from the cassava hornworm cadavers is a betabaculovirus designated Erinnyis ello granulovirus (ErelGV). The 102,759 bp long genome has a G + C content of 38.7%. We found 130 putative ORFs coding for polypeptides of at least 50 amino acid residues. Only eight genes were found to be unique. ErelGV is closely related to ChocGV and PiraGV isolates. We did not find typical homologous regions and cathepsin and chitinase homologous genes are lacked. The presence of he65 and p43 homologous genes suggests horizontal gene transfer from Alphabaculovirus. Moreover, we found a nucleotide metabolism-related gene and two genes that could be acquired probably from Densovirus. CONCLUSIONS: The ErelGV represents a new virus species from the genus Betabaculovirus and is the closest relative of ChocGV. It contains a dUTPase-like, a he65-like, p43-like genes, which are also found in several other alpha- and betabaculovirus genomes, and two Densovirus-related genes. Importantly, recombination events between insect viruses from unrelated families and genera might drive baculovirus genomic evolution.

Lymantria dispar iflavirus 1 (LdIV1), a new model to study iflaviral persistence in lepidopterans.

The cell line IPLB-LD-652Y, derived from the gypsy moth (Lymantria dispar L.), is routinely used to study interactions between viruses and insect hosts. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered to persistently infect IPLB-LD-652Y. LdIV1 belongs to the genus Iflavirus. LdIV1 formed icosahedral particles of approx. 30 nm in diameter and contained a 10, 044 nt polyadenylated, positive-sense RNA genome encoding a predicted polyprotein of 2980 aa. LdIV1 was induced by a viral suppressor of RNA silencing, suggesting that acute infection is restricted by RNA interference (RNAi). We detected LdIV1 in all tested tissues of gypsy-moth larvae and adults, but the virus was absent from other L. dispar-derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group of viruses, many of which covertly infect their hosts.

Detection and quantitation of Agrotis baculoviruses in mixed infections.

At least four distinct baculoviruses, namely the Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), the A. segetum nucleopolyhedrovirus B (AgseNPV-B), the Agrotis ipsilon nucleopolyhedrovirus (AgipNPV) and the A. segetum granulovirus (AgseGV) have been isolated from larval stages (cutworms) of the species A. segetum and A. ipsilon (Lepidoptera: Noctuidae), which are serious soil pests in agriculture. Cutworms can become infected by at least one of these four baculoviruses and also co-infections of A. segetum larvae with AgseNPV-B and AgseGV are observed under laboratory conditions. Because of their adaption to common hosts and the occurrence in mixed infections, these viruses have a considerable potential as biological control agents of cutworms and are suitable objects to decipher the co-evolution and population dynamics of baculoviruses in mixed infections. However, to facilitate studies on these viruses a reliable tool for detection and identification is essential. A method based on highly specific oligonucleotide primers for multiplex polymerase chain reaction (PCR) that led to the amplification of discriminating fragments of the polyhedrin (polh) and granulin (gran) gene of AgseNPV-A, AgseNPV-B, AgipNPV and AgseGV, was established. Furthermore, the AgseNPV-B and AgseGV specific pairs of primers were applied in real-time PCR (qPCR) for AgseNPV-B/AgseGV ratio determination in samples of mixed infections. It is demonstrated further that for quantifying NPVs and GVs in mixed infections, the method of occlusion body isolation is most crucial and significantly influences the results.

Histopathology of cotton bollworm midgut infected with Helicoverpa armigera cytoplasmic polyhedrosis virus

This research was carried out to examine cytopathological effects of Helicoverpa armigera Cytoplasmic polyhedrosis virus (HaCPV) on infected midgut cotton bollworm (Helicoverpa armigera) using transmission and scanning electron microscope. The symptoms on infected host larvae of the host, compared with healthy ones, were getting swollen with milky-white and fragile Histopathological examinations showed infection with HaCPV small polyhedral inclusion bodies (PIB) after 1 or 2 days which were observed in columnar cells of midgut. Virions were partially or completely occupied in a polyhedral matrix to form polyhedral inclusion bodies (PIB) at periphery of virogenic stroma. PIBs were measured 0.5 to 3.5 mm and virions about 46 nm in diameter. Microvilli of infected columnar cells were affected and degenerated immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 3 days after infection. In addition,PIB were found in goblet cells, 5 or 6 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organells were left. These cytopathic effects caused in the midgut by HaCPV on cotton bollworm larvae are essentially similar to those have been reported for lepidoperan and dipteran infection by CPV.