Endogenous retroviruses related to jaagsiekte sheep retrovirus.

Ovine betaretroviruses consist of exogenous viruses [jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus, (ENTV)] associated with neoplastic diseases of the respiratory tract and 15-20 endogenous viruses (enJSRV) stably integrated in the ovine and caprine genome. Phylogenetic analysis of this group of retroviruses suggests that the enJSRV can be considered as 'modern' endogenous retroviruses with active, exogenous counterparts. Sequence analysis of JSRV, ENTV and enJSRV suggests that enJSRV do not directly contribute to the pathogenesis of ovine pulmonary adenocarcinoma (OPA) or enzootic nasal tumor through large-scale recombination events, but small-scale recombination or complementation of gene function cannot be excluded; experiments involving enJSRV-free sheep, which have not been found, would be needed to investigate this possibility. Evidence of expression of enJSRV structural proteins in tissues of the reproductive tract and lung implies that they do not have a primary role in disease. However, experimental exploitation of exogenous/endogenous retrovirus sequence differences by producing chimeras has been useful in establishing the determinants of JSRV Env-induced transformation. Even if enJSRV do not have a direct role in OPA, their expression during ontogeny or in neonatal life may impact the likelihood of exogenous JSRV infection and disease outcome via the induction of immunological tolerance. Aside from any role in disease, enJSRV loci may serve as useful genetic markers in the sheep and their frequent expression in the reproductive tract of the ewe may portend an important physiologic role in sheep.

Molecular cloning and characterization of a metal responsive Chironomus tentans alpha-tubulin cDNA.

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Sensitive molecular biomarkers of metal exposure are sorely needed. We have isolated a cDNA from the midge Chironomus tentans that is transcribed in all organs and developmental stages. The cDNA encodes a protein, designated Chironomus tentans alpha-tubulin 1 (CTTUB1), which has significant similarities with invertebrate and vertebrate alpha-tubulins. CTTUB1 is abundantly transcribed in embryos and to a lesser extent in adults and larvae. CTTUB1 RNA and protein abundances are increased in larvae exposed to copper or cadmium. The pattern of cellular distribution of CTTUB1 protein in the midgut epithelial cells was radically affected by cadmium. In the midgut cells of unexposed larvae, CTTUB1 was found evenly distributed throughout the cytoplasm, while in cadmium-exposed larvae, CTTUB1 was mostly concentrated along the basolateral plasma membrane. A mechanism for the regulation of alpha-tubulin synthesis by cadmium is proposed. This is the first report on the isolation of a metal responsive gene from a neartic aquatic insect.

Sindbis virus-induced silencing of dengue viruses in mosquitoes.

Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN-2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN-2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN-derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN-2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod-borne viruses in mosquito vectors, studying vector-virus interactions, and silencing endogenous mosquito genes.

Jaagsiekte sheep retrovirus proviral clone JSRV(JS7), derived from the JS7 lung tumor cell line, induces ovine pulmonary carcinoma and is integrated into the surfactant protein A gene.

Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV(JS7)) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV(JS7) is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

Expression of human MxA protein in mosquito cells interferes with LaCrosse virus replication.

Human MxA protein inhibits LaCrosse virus (LAC virus; family Bunyaviridae) replication in vertebrate cells and MxA-transgenic mice. LAC virus is transmitted to humans by Aedes triseriatus mosquitoes. In this report, we have shown that transfected mosquito cells expressing the human MxA cDNA are resistant to LAC virus but permissive for Sindbis virus (family Togaviridae) infection.

Characterization of the structural gene promoter of Aedes aegypti densovirus.

Aedes aegypti densonucleosis virus (AeDNV) has two promoters that have been shown to be active by reporter gene expression analysis (B. N. Afanasiev, Y. V. Koslov, J. O. Carlson, and B. J. Beaty, Exp. Parasitol. 79:322-339, 1994). Northern blot analysis of cells infected with AeDNV revealed two transcripts 1,200 and 3,500 nucleotides in length that are assumed to express the structural protein (VP) gene and nonstructural protein genes, respectively. Primer extension was used to map the transcriptional start site of the structural protein gene. Surprisingly, the structural protein gene transcript began at an initiator consensus sequence, CAGT, 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promoter. Constructs with the beta-galactosidase gene fused to the structural protein gene were used to determine elements necessary for promoter function. Deletion or mutation of the initiator sequence, CAGT, reduced protein expression by 93%, whereas mutation of the TATAA sequence at map unit 61 had little effect. An additional open reading frame was observed upstream of the structural protein gene that can express beta-galactosidase at a low level (20% of that of VP fusions). Expression of the AeDNV structural protein gene was shown to be stimulated by the major nonstructural protein NS1 (Afanasiev et al., Exp. parasitol., 1994). To determine the sequences required for transactivation, expression of structural protein gene-beta-galactosidase gene fusion constructs differing in AeDNV genome content was measured with and without NS1. The presence of NS1 led to an 8- to 10-fold increase in expression when either genomic end was present, compared to a 2-fold increase with a construct lacking the genomic ends. An even higher (37-fold) increase in expression occurred with both genomic ends present; however, this was in part due to template replication as shown by Southern blot analysis. These data indicate the location and importance of various elements necessary for efficient protein expression and transactivation from the structural protein gene promoter of AeDNV.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae.

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322-339; Afanasiev et al. (1999) Virology 257: 62-72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139-159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev etal., 1999; Allen-Muira et al. (1999) Virology 257: 54-61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10-0.15 M inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA.

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine-rich domains, two of which flank a proline/serine/threonine-rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal-responsive gene from an aquatic insect.

Development of a Sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of Aedes aegypti following per os infection.

A double subgenomic Sindbis (dsSIN) virus, MRE/3'2 J/GFP, was constructed to efficiently express green fluorescent protein (GFP) in the midgut of Aedes aegypti following per os infection. The MRE/3'2 J/GFP RNA genome contained the nonstructural genes and cis-acting sequences of the dsSIN virus, TE/3'2 J/GFP, but had the structural genes of MRE16 SIN virus. MRE/3'2 J/GFP virus, unlike TE/3'2 J/GFP virus, efficiently infected mosquitoes orally. At 1-2 days postinfection, GFP was observed as multiple foci of expression on the lumenal side of the midgut. At 10-12 days postinfection, thirteen of fifteen mosquitoes infected with MRE/3'2 J/GFP virus had high levels of GFP expression in the mosquito midgut. The MRE3'2 J dsSIN expression system should be an important tool for efficient gene expression in Ae. aegypti midguts.

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor.

Ovine pulmonary carcinoma, a contagious lung cancer of sheep, is caused by the oncogenic jaagsiekte sheep retrovirus (JSRV) that is closely related to a family of endogenous sheep retroviral sequences (ESRVs). By using exogenous virus-specific U3 oligonucleotide primers, the entire JSRV proviral genome or its 3' part was amplified from tumor DNA. Analysis of these proviral sequences revealed a novel open reading frame (ORF) within the pol coding region, designated ORF X, which was well conserved in ESRV and JSRV sequences. Deduced amino acids of ORF X showed similarity to a portion of the mammalian adenosine receptor subtype 3, a member of the G-protein-coupled receptor family. Comparison of deduced env amino acids of six JSRV strains from three continents identified 15 residues that defined two distinct genotypes of JSRVs. Sequence analysis identified two highly variable regions between JSRV and ESRV in the transmembrane domain of env (TM) and the 3' unique sequence (U3) of the long terminal repeat, from which JSRV-specific DNA probes were derived. By using these DNA probes in Southern hybridization, for the first time we successfully identified JSRV proviral sequences in tumor genomic DNA in the presence of multiple ESRV loci, validating the use of exogenous virus-specific DNA probes in the analysis of oncogenic proviral integration sites and identification of integrated exogenous proviral sequences.

Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system.

Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins.

Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus.

Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses.

Molecular and genetic ecotoxicologic approaches to aquatic environmental bioreporting.

Molecular and population genetic ecotoxicologic approaches are being developed for the utilization of arthropods as bioreporters of heavy metal mixtures in the environment. The explosion of knowledge in molecular biology, molecular genetics, and biotechnology provides an unparalleled opportunity to use arthropods as bioreporter organisms. Interspecific differences in aquatic arthropod populations have been previously demonstrated in response to heavy metal insult in the Arkansas River (AR) California Gulch Superfund site (CGSS). Population genetic analyses were conducted on the mayfly Baetis tricaudatus. Genetic polymorphisms were detected in polymerase chain reaction amplified 16S mitochondrial rDNA (a selectively neutral gene) of B tricaudatus using single-strand conformation polymorphism analysis. Genetic differences may have resulted from impediments to gene flow in the population caused by mortality arising from exposure to heavy metal mixture pollution. In laboratory studies a candidate metal-responsive mucinlike gene, which is metal and dose specific, has been identified in Chironomus tentans and other potential AR-CGSS bioreporter species. Population genetic analyses using the mucinlike gene may provide insight into the role of this selectable gene in determining the breeding structure of B. tricaudatus in the AR-CGSS and may provide mechanistic insight into determinants of aquatic arthropod response to heavy metal insult. Metal-responsive (MR) genes and regulatory sequences are being isolated, characterized, and assayed for differential gene expression in response to heavy metal mixture pollution in the AR-CGSS. Identified promoter sequences can then be engineered into previously developed MR constructs to provide sensitive in vitro assays for environmental bioreporting of heavy metal mixtures. The results of the population genetic studies are being entered into an AR geographic information system that contains substantial biological, chemical, and geophysical information. Integrated spatial, structural, and temporal analyses of these parameters will provide invaluable information concerning environmental determinants that restrict or promote gene flow in bioreporter populations.

Use of the Sindbis replicon system for expression of LaCrosse virus envelope proteins in mosquito cells.

The Sindbis replicon expression system was used to express La Crosse (LAC) virus envelope glycoprotein genes in both mammalian and mosquito cell culture. Replicon expressed LAC proteins had correct molecular mass (Mr) and were antigenically similar to wild type LAC envelope proteins. In addition, LAC G1 and G2 proteins colocalized when expressed from separate constructs in both mammalian and mosquito cells suggesting that they were trafficked through the cell similarly to wild type LAC proteins. A truncated form of the G1 protein was secreted from mosquito cells when expressed alone. The truncated G1 protein was also secreted from mosquito cells when expressed with the G2 protein, but to a lesser extent than when expressed alone, suggesting that the G2 protein sequestered G1 protein intracellularly. The Sindbis replicon system is a powerful tool for the study of LAC virus protein maturation within mosquito cells and mosquitoes.

Gene expression and regulation from the p7 promoter of Aedes densonucleosis virus.

The nonstructural proteins NS1 and NS2 are thought to be expressed from the p7 promoter of Aedes densonucleosis virus (AeDNV). To study gene expression from the p7 promoter, eight different plasmids were constructed by fusing beta-galactosidase or beta-glucuronidase into the genome so that the reporter gene was in different open reading frames and under the transcriptional control of the p7 promoter. After transfection into C6/36 Aedes albopictus cells, constructs generated comparable amounts of RNA, but only the NS1 and NS2 fusion constructs produced appreciable levels of active enzyme. NS1 and NS2 fusion constructs contained wild-type AeDNV sequences from the p7 promoter downstream to nucleotide 458. The remaining constructs, with the exception of p7GUS.rf3, lacked some or all of these necessary sequences and inefficiently produced protein. These data suggest that sequences downstream of the p7 promoter play a role in translational regulation of gene expression from the p7 promoter of AeDNV.

Development of a chimeric sindbis virus with enhanced per Os infection of Aedes aegypti.

The TE/3'2J double subgenomic Sindbis (dsSIN) viruses have been used to stably express genes in Aedes aegypti nerve and salivary gland tissues. However, because these viruses inefficiently infect Ae. aegypti when administered by the per os route, TE/3'2J viruses must be intrathoracically inoculated into the mosquitoes to infect these tissues. A Malaysian Sindbis (SIN) virus isolate (MRE16) does efficiently infect Ae. aegypti midgut tissues after ingestion, and approximately 95% of these mosquitoes also develop disseminated infections within 14 days. We have sequenced the entire 26S RNA of MRE16 virus and have developed a chimeric SIN cDNA infectious clone, designated MRE1001, which contains sequence elements of TE/3'2J and MRE16 virus. MRE1001 virus efficiently infects midgut cells, and greater than 90% of infected mosquitoes develop disseminated infections after 14 days extrinsic incubation. The chimeric MRE1001 cDNA clone should allow identification of viral determinants of midgut infection and dissemination and lead to the development of new SIN virus expression systems.

Effect of heavy metals on Aedes aegypti (Diptera: Culicidae) larvae.

Studies were conducted to determine the biological effects of heavy metals on the development of Aedes aegypti. Embryos immersed in 32 ppm Cu or 5 ppm Cd did not hatch. The arrest of hatching was in part reversible by removal of the heavy metals. The mortality rate of third-instar larvae exposed to heavy metals for 24 h was metal and dose dependent; the 50% lethal concentration (LC50) endpoints were 3.1, 16.5, and 33 ppm for Hg, Cd, and Cu, respectively. Interestingly, a proportion of Aedes aegypti third-instar larvae exposed to either Cu or Cd for 24 h failed to produce a dissectable peritrophic matrix. This failure to produce a dissectable peritrophic matrix also was metal and dose dependent. These results are discussed in the context of Aedes aegypti as a model system for investigating the molecular biological effects of heavy metals in aquatic insects.

Envelope glycoprotein nucleotide sequence and genetic characterization of North American ovine lentiviruses.

Ovine lentiviruses (OvLV) resemble human immunodeficiency viruses in genomic organization, viral heterogeneity, and spectrum of cytophenotypic expression. To gain a better understanding of the relationship of North American OvLV isolates with other characterized OvLV strains, the complete DNA nucleotide sequence of the env region of a highly lytic (rapid/high) OvLV strain (85/34) was determined and compared with the sequence of amplicons within env of three other OvLV strains of varying cytophenotype and isolated from the same flock of sheep. LTR and pol regions also were compared among these strains. The env region of 85/34 was 986 codons in length and the reported nucleotide sequence showed features shared by other OvLV including heavy glycosylation and conserved and hypervariable regions within the surface membrane protein region. Phylogenetic analyses of regions within LTR, reverse transcriptase, and env grouped the four virus strains together and similar to the maedi-visna OvLV strains, including visna virus, South African ovine maedi visna virus, and EV1 (British OvLV isolate), but they were distinct from caprine arthritis encephalitis virus.

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).