Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay

Summary.  The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10⁵-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control. Received April 12, 2000 Accepted September 25, 2000     

First complete and productive cell culture model for members of the genus Iridovirus

Chilo iridescent virus (CIV; the type strain of the genus Iridovirus) replicates productively in larvae of the boll weevil, Anthonomus grandis. This study focuses on characterizing productive infections of a boll weevil cell line, BRL-AG-3A (AG3A), starting with CIV reared in the waxworm, Galleria mellonella. We show that CIV can be continually and productively passaged to high titer in AG3A cells. The replication of larval-derived CIV in AG3A was analyzed by observing viral DNA replication and restriction endonuclease digestion profiles, morphogenesis, and infectivity using TCID₅₀ assays with AG3A as an indicator cell line. The data showed that virus passaged in the AG3A host is stable. AG3A cells are more efficient than previously utilized CF-124T cells from Choristoneura fumiferana. This system constitutes a superior model for cellular and molecular studies on CIV; it represents the first complete, productive cell culture model for the replication of CIV or any member of the genus Iridovirus.     

Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV(XS); 400 microg/ml), UV-irradiated virus (CIV(UV); 10 microg/ml) and CVPE (CIV protein extract; 10 microg/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 microg/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV(UV) or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV(UV) particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV(UV), CIV(XS) or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae, apoptosis: (i) requires entry and endocytosis of virions or virion proteins, (ii) is inhibited under conditions permitting early viral expression, and (iii) requires the JNK signaling pathway. This is the first report of JNK signal requirement during apoptosis induction by an insect virus.     

Molecular Anatomy of Chilo Iridescent Virus Genome and the Evolution of Viral Genes

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.     

Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan,South China Sea and Southeast Asian countries

Summary.  Tropical iridovirus infection causes severe epizootic resulting in mass mortalities and large economic losses in freshwater ornamental fishes cultured in Southeast Asian countries, in wild fish seedlings captured in South China Sea, and in marine fishes farmed in Japan, Singapore, and Thailand. All of tropical iridovirus-infected fishes histopathologically showed the systemic formation of inclusion body-bearing cells and necrosis of virus-infected splenocytes and hematopoietic cells. We designed primer sets for the ATPase gene and the major capsid protein (MCP) gene and sequenced the PCR products derived from 5 iridovirus isolates from sea bass in South China Sea, red sea bream in Japan, brown-spotted grouper with a grouper sleepy disease in Thailand, dwarf gourami from Malaysia and African lampeye from Sumatra Island, Indonesia. The ATPase gene and the MCP gene of these 5 viral isolates were highly homologous (> 95.8%, > 94.9% identity, respectively) and the deduced amino acid sequences of the ATPase and the MCP were also highly identical (> 98.1%, > 97.2% identity, respectively). Based on the high homology, these 5 isolates of tropical iridovirus from various fishes in geographically different regions were determined to have a single origin and to be native to Southeast Asian regions. However, these sequences were far different from those of members of the genera Ranavirus, Lymphocystivirus and Iridovirus in the Family Iridoviridae. We propose a new genus “Tropivirus” for tropical iridovirus in the Family Iridoviridae. Received October 16, 2001; accepted July 5, 2002     

Cladistic analysis of iridoviruses based on protein and DNA sequences

Summary. Cladograms of iridoviruses were inferred from bootstrap analysis of molecular data sets comprising all published protein and DNA sequences of the major capsid protein, ATPase and DNA polymerase genes of members of the Iridoviridae family Iridovirus. All data sets yielded cladograms supporting the separation of the Iridovirus, Ranavirus and Lymphocystivirus genera, and the cladogram based on data derived from major capsid proteins further divided both the Iridovirus and Ranavirus genera into two groups. Tests of alternative hypotheses of topological constraints were also performed to further investigate relationships between infectious spleen and kidney necrosis virus (ISKNV), an unclassified fish iridovirus for which the complete genome sequence data is available, and other iridoviruses. Cladograms inferred and results of Shimodaira–Hasegawa tests indicated that ISKNV is more closely related to the Ranavirus genus than it is to the other genera of the family.Received November 21, 2002; accepted June 9, 2003 Published online August 18, 2003     

Sequence comparison of the major capsid protein gene from 18 diverse iridoviruses

Summary.  Insect iridoviruses (IV) have been found on all continents of the world and in a broad range of insect hosts. The host range for a single strain can cross several insect orders. This along with a paucity of molecular information on all but a few members has led to confusion in the taxonomy and classification of these viruses and in the identification of potentially novel isolates. To address this problem consensus PCR primers were designed to amplify and sequence a 500 bp region of the major capsid protein (MCP) gene. PCR products were amplified from eighteen IVs belonging to the genus Iridovirus. No product was observed for the chloriridovirus IV3. Phylogenetic analysis of the partial MCP gene sequence showed that the iridovirus genus can be divided into three groups. These results support previous studies where a range of molecular techniques were used. Group I contained PjIV and IV31, group II contained IV6 (CIV), IV21, and IV28, and group III contained IV1 (TIV), IV2 (SIV), IV9 (WIV), IV10, IV16, (CzIV), IV18, IV22, IV23 (BbIV), IV24, IV29, IV30, AgIV and an un- described weevil IV. There was no correlation of relatedness with host of isolation but there was some correlation with geographic region of isolation. Sequence analysis also raised issues concerning the purity of some virus stocks and supported the view that some isolates should be considered as variants of one virus species. Received February 10, 1998 Accepted May 13, 1998     

Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus

Summary. In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented.     

Identification and characterization of a novel capsid protein encoded by Singapore grouper iridovirus ORF038L

Singapore grouper iridovirus (SGIV) is an important pathogen isolated from grouper, Epinephelus tauvina, and characterized as a novel ranavirus. To better understand the function of viral structural genes involved in SGIV infection and virus–host interactions, a candidate gene, VP38 (ORF038L), was investigated in this study. SGIV VP38 was found to encode a 170-aa peptide containing an RGD motif, and it showed significant identity only to members of the genus Iridovirus, family Iridoviridae, except megalocytivirus. The VP38 gene was identified by temporal expression pattern analysis and drug inhibition assay as a late (L) gene. Immunofluorescence localization revealed that P38 was distributed predominately in the cytoplasm and that association of VP38 with viral factories increased at the late stage of SGIV infection. Consistent results from immunoelectron microscopy (IEM) and western blot analysis revealed that SGIV VP38 is a viral capsid protein. Furthermore, antibodies specific for SGIV VP38 exhibited substantial SGIV-neutralizing activity in vitro, suggesting that VP38 might play an important role in SGIV infectivity.     

A comparison of the structural polypeptides of three iridescent viruses (types 6, 9, and 16) and the mapping of the DNA region coding for their major capsid polypeptides

Summary The iridoviruses fromWiseana cervinata (WIV, type 9),Costelytra zealandica (CzIV, type 16) andChilo suppressalis (CIV, type 6) were compared by SDS-PAGE and Western protein blotting for antigenic determinants. The major capsid proteins were isolated and oligonucleotide probes were synthesized from the partial amino acid sequences. The DNA regions coding for the major capsid proteins of WIV (VP52), CzIV (VP53) and CIV (VP50) were located by hybridization of the oligonucleotide probes to blots of the viral DNA. The major capsid protein was used as the zero point for the proposed linearized maps of these viruses.Using antibody and¹²⁵I-labelling, several proteins were identified as being on the surface of the virion. It was also shown that CIV was not as antigenically distinct from these two viruses as previously reported.     

A tymovirus from Calopogonium mucunoides in Malaysia is not clitoria yellow vein tymovirus

Summary. A tymovirus isolated from Malaysian crops of Calopogonium mucunoides has been shown to have virions that are serologically indistinguishable from those of clitoria yellow vein tymovirus. We have sequenced the virion protein (VP) gene of the virus and have found that although it is a member of the cluster that includes CYVV, it is the most distinct member of that cluster (< 62% sequence identity with all the others), and is clearly a separate species, which we propose should be named calopogonium yellow vein virus. Most of the serological specificity of the virions of tymoviruses seems to reside in the C-terminal hexapeptide of the virion protein. Received December 12, 1996 Accepted March 7, 1997     

<Emphasis Type="Italic">Rana grylio</Emphasis> virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK–GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells. The sequence reported here has been deposited in the GenBank database under accession no EU747722.     

Development and infectivity of eggs and larvae derived from pigs trickle-infected with Oesophagostomum dentatum at different dose levels

We examined the impact of different Oesophagostomum dentatum dose levels and durations of infection on the development and infectivity of the following generation. Pigs were trickle-infected with 200, 2,000 or 20,000 L₃/week over 20 weeks. Egg hatch assays were performed at monthly intervals; however, no consistent differences were found between any of the dose groups in the development of eggs into first-stage larvae. To compare larval infectivity, larvae were derived from faecal cultures set up from the low- and the high-dose groups in the early and the late part of the experiment, and were inoculated into helminth-free pigs (5,000 L₃/pig). Worm establishments were significantly higher (P < 0.05) in the group of pigs receiving larvae derived early in the experiment from the low-dose group compared with the two groups receiving larvae from high-dose groups, thus indicating an adverse effect of high doses of trickle infection on the later infectivity of L₃ larvae derived from excreted eggs. Received: 16 March 1998 / Accepted: 15 June 1998     

Inhibition of macromolecular synthesis in cells infected with an Invertebrate Virus (iridovirus type 6 or CIV)

Summary Chilo Iridescent Virus (CIV), an invertebrate virus, rapidly inhibits cellular RNA, DNA and protein synthesis in permissive and non permissive vertebrate and invertebrate cell lines. The integrity of the viral genome is not required for inhibitory expression, since viral proteins solubilized from CIV by freezing and treatment with EDTA exhibit inhibitory properties similar to those of intact virions.With 3 Figures     

Expression of vaccinia virion surface tubule protein as a virus specific cell surface antigen

Summary The sequential expression of a vaccinia virus specific antigen on the surface of infected cells has been followed by¹²⁵I-labelled anti-vaccinia IgG. After an initial drop (during the first 30 minutes of infection) the amount of viral antigen at the cell surface increased steadily for the 12 hours tested. The expression of the antigen was found to depend on protein and RNA synthesis from the start, but dependent on DNA synthesis only after 4 hours. The sensitivity of the phenomenon to ultraviolet light irradiation of the virus suggests that the genetic information needed for the expression of the antigen resides in the viral genome. The antigen has been identified as the virion surface tubule, a tubule-like structure on the surface of the intact virion. It is known that vaccinia virus infection of cells starts with the fusion of the virion envelope with the host plasma membrane.It is here proposed that initially tubule from input virus is detected as viral antigen on the cell surface. Subsequently, virus tubule protein synthesisedde novo migrates and is detectable as the virus specific cell surface antigen.With 3 Figures     

Characterization of Apoptotic Activities During Chlamydia trachomatis Infection in Primary Cervical Epithelial Cells

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P?<?0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p?<?0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.     

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations

Summary Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV₃) and chilo iridescent virus (CIV), two other well known viruses of the familyIridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A₂. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.     

Investigation into the Anti-inflammatory and Antigranuloma Activity of <Emphasis Type="Italic">Colchicum luteum</Emphasis> Baker in Experimental Models

The present study evaluates the anti-inflammatory and antigranuloma activity of CLHE in experimental models, viz. carrageenan-induced paw edema, subcutaneous cotton pellet implantation-induced granuloma formation, and complete Freund's adjuvant-induced stimulation of peritoneal macrophages in rats. Serum TNF-alpha, IL-6, and IL-1 beta levels were estimated as markers for global effects of inflammation. TNF-R1 protein expression was estimated in stimulated peritoneal macrophages. There was a significant reduction (P < 0.05) of paw edema in the CLHE-treated groups as compared to control. In the cotton pellet-induced granuloma model, there was a significant (P < 0.05) reduction in the dry granuloma weight and serum TNF-alpha, IL-6, and IL-1beta levels in the CLHE-treated group as compared to control. Immunoblot analysis for TNF-R1 also demonstrated a significant reduction in the receptor protein expression on stimulated macrophages. Result of the present study thus demonstrates and validates the antigranuloma activity of CLHE.