Progress 08/01/07 to 12/31/11
Outputs
OUTPUTS: The MdHV, described initially as a non-occluded, enveloped, rod-shaped, double-stranded DNA virus, is responsible for the salivary gland hypertrophy symptoms observed in feral populations of adult house flies. Infection by this virus sterilizes female house flies, since adults displaying symptoms of SGH show no sign of ovarian development. Utilizing Nycodenz gradients, MdHV was purified from hypertrophic glands of infected house flies and its genome sequenced. Putative open-reading frames were tested with RT-PCR and RACE protocols producing a validated transcriptome for this virus. Both nucleocapsids and enveloped virions were present in salivary gland cells. In contrast, thin sections of midguts, ovaries, abdominal fat body, crops, air sacs and brains showed the presence of enveloped virions in vacuoles of tracheal cells associated with these organs. In Stomoxys calcitrans, an insect that does not support SGH symptoms, the MdHV was shown to replicate in adult tissues and inhibit egg development in challenged flies. Comparative TEM analysis demonstrated that the replicative strategy displayed by MdHV was distinct from that observed with the GpHV in the salivary gland tissue. A series of monospecific polyclonal antibodies were prepared from proteins in recombinant systems expressing the structural and nonstructural MdHV ORFs; the only antibody capable of neutralizing infectivity were those targeting the intact virus or the major ORF 96 envelope protein. During field surveys, MdHV has been detected in house fly samples from North America, Europe, Asia, the Caribbean, and the Southwestern Pacific. Nucleotide sequences from 16 different geographic isolates were highly homologous, and detected polymorphism was correlated with geographic source. When challenged with various isolates, 24h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. This resistance could be broken by pretreatment of flies with a reducing agent, potentially by disrupting the peritrophic matrix and facilitating viral passage into the hemocoel. Delivery strategies of the MdSGHV as a biological control agent were examined by comparing oral and topical treatments (Potter spray tower, immersion) of house flies with viral suspensions. The most effective virus administration was a brief immersion in a homogenate of infected flies, resulting in 50% infection in resistant flies. Disease rates transmitted from virus-infected to healthy flies in small populations of 50 or 100 flies ranged from an average 3% to 24% and did not vary between three tested isolates that originated from different continents. Introduction of an initial proportion of 40% infected flies into fly populations did not result in epizootics. Instead, long-term observations demonstrated that MdSGHV-infection levels declined over time resulting in a 10% infection rate after passing through ten filial generations. In all experiments, induced disease rates were significantly higher in male flies than in female flies and might be explained by male-specific behaviors that increased contact with viremic flies and/or virus-contaminated surfaces. PARTICIPANTS: D. Boucias-PI University of Florida Gainesville FL V. Lietze-CoPI University of Florida Gainesville FL C Geden-CoPI USDA/ARS Gainesville FL P. Prompiboon (post doc) T. Salem (postdoc) A. Garcia-Maruniak (cooperator) Agencies and other laboratories: A. Abd-Alla, A. Parker, and M Vreysen- International Atomic Energy Agency (Siebersdorf, Austria) J. M. Vlak, Wageningen University, The Netherlands M. Bergoin, University Montpellier, France J. P. Burand, University of Massachusetts, Amherst J. A. Jehle, DLR Rheinpfalz, Germany TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This MdHV, characterized as a sterilizing virus of house flies, was subjected to a three-year examination by our research group. The results of our studies on this novel virus were reported in 16 scientific manuscripts including an research overview in the Annula Review of Entomology. To date an extensive collection of MdHV strains has been compiled and partially characerized. The ~125 kbp genome of the MDHV has been completed and annotated and open-reading frames encoded for by the MdSGHV have been validated as have the UTR regions and poly-A processing regions. Comparative analyses with the research group at the IAEA has led to the formation of a new virus family-the Hytrosavirus (HV) of which the MdHV is placed within the genus Muscavirus thus the reference MdHV. A series of universal primers have been developed that can be used to detect new members belonging to this family. These primers will allow the community to detect related hytrosviruses in other insect species. Bioassays and histologicsl studies demonstrated that this virus in addition to replicating in the salivary gland could replicate in other somatic tissues of the house fly and other related fly species. Viral replication appeared to infect additional gland tissues potentially disrupting hormone levels resulting in the observed block in vitellogenesis resulting form a down-regulation of transcription in the fat body. Resistance to oral infection in adult house flies was associated with the maturation of the adult peritrophic membrane; treatment with various reducing agents disrupted this barrier and dramatically increased the susceptibility of flies to viremia. In-depth electron microscopy and immunoelectron microscopy has revealed that the replicative pathway of this virus has many steps that are unique from what is observed in other dsDNA viruses. Simulated field releases made in large cages revealed that this virus can be transmitted to older flies and once introduced is maintained over many generations at rates comparable to what is observed in feral populations.
Publications
- Lietze, Verena-Ulrike, Christopher J. Geden, Melissa Doyle, and Drion G. Boucias 2012. Transmission dynamics and persistence of MdSGHV in laboratory house fly (Musca domestica) populations. Appl. Environ. Microbiol. 78(2):311-317.
- Lietze, V.-U., K. Sims, T.Z. Salem, C.J. Geden, and D.G. Boucias. 2009. Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), via salivary secretions and excreta. Journal of Invertebrate Pathology 101: 49-55.
- Salem, T.Z., A. Garcia-Maruniak, V.-U. Lietze, J.E. Maruniak, and D.G. Boucias. 2009. Analysis of transcripts from predicted ORFs of the Musca domestica salivary gland hypertrophy virus (MdSGHV). Journal of General Virology 90: 1270-1280.
- Abd-Alla, A.M.M., J.M. Vlak, M. Bergoin, J.E. Maruniak, A. Parker, J.P. Burand, J.A. Jehle, and D.G. Boucias. 2009. Hytrosaviridae: A proposal for classification and nomenclature of a new insect virus family. Archives of Virology 154: 909-918.
- Garcia-Maruniak, A., A.M.M. Abd-Alla, T.Z. Salem, A.G. Parker, V. Lietze, M.M. van Oers, J.E. Maruniak, W. Kim, J.P. Burand, F. Cousserans, A.S. Robinson, J.M. Vlak, M. Bergoin, and D.G. Boucias. 2009. Comparative analysis of two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica. Journal of General Virology 90: 334-346.
- Garcia-Maruniak, A., J.E. Maruniak, W. Farmerie, and D.G. Boucias. 2008. Sequence analysis of a non-classified, non-occluded DNA virus that causes salivary gland hypertrophy of Musca domestica, MdSGHV. Virology 377: 184-196.
- Geden, C.J., V. Lietze, and D.G. Boucias. 2008. Seasonal prevalence and transmission of salivary gland hyperplasia virus of house flies, Musca domestica L. (Diptera: Muscidae). Journal of Medical Entomology 45 (1): 42-51.
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Progress 08/01/10 to 07/31/11
Outputs
OUTPUTS: MdSGHV has been detected in house fly samples from North America, Europe, Asia, the Caribbean, and the Southwestern Pacific. Nucleotide sequences from 16 different geographic isolates were highly homologous, and detected polymorphism was correlated with geographic source. With all isolates, 24h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. Regardless of the MdSGHV isolate tested, all susceptible insects displayed similar degrees of SGH and a complete suppression of oogenesis. House flies (Musca domestica L.) infected with MdSGHV were found in fly populations collected from 12 out of 18 Danish livestock farms. Infection rates ranged from 0.5 to 5 and averaged 1.2 percent overall. None of the stable flies, rat-tail maggot flies or yellow dung flies collected from virus-positive farms displayed SGH. In laboratory transmission tests, no SGH symptoms were observed in heterologous fly hosts injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), both injection and topical application of MdSGHV resulted in suppression of ovarian development similar to that observed in challenged house flies. The tissue tropism of Musca domestica salivary gland hypertrophy virus (MdSGHV) infecting adult house flies was examined by transmission electron microscopy (TEM) and quantitative real-time PCR. The midgut, ovary, abdominal fat body, crop, air sac and brain showed the presence of enveloped virions in vacuoles of associated tracheal cells. No sites of viral morphogenesis were detected in the tracheal cells. Analysis of MdSGHV DNA and transcript titers revealed that viral DNA and viral transcripts was present in all tissue samples collected from MdSGHV-infected flies; the highest levels in salivary glands and at the lowest levels in hemolymph. However, over the range of different tissues that were analyzed, there was no correlation between estimated quantities of genome copies and viral transcripts. The function of viral transcripts in host tissues that do not show sites of viral morphogenesis remains to be elucidated. Based on the complete genome sequence of Glossina pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from e66-ODV homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47C. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The MdSGHV has been defined as a unique virus that is globally distributed. Work done on this virus and on those viruses infecting the tsetse fly has led to the description of a new virus family Hytrosaviridae that contains the genera Glossinavirus and Muscavirus. During this past year we have developed an EST data base on infected/healthy flies that has provided a framework to address the host/virus interactions. The recognition that this virus replicates in non salivary gland tissues is important in elucidating the mechanism underlying the virus induced sterility. Ongoing research is addressing the impact of this virus on the corpus cardica and JH biosynthesis. The development of universal primers for SGHVs provides a tool for detecting hytrosaviruses in tsetse flies and house flies. The availability of these primers will facilitate detection of hytrosaviruses in a wide range of insects and is expected to result in the discovery of other hytroviruses in other insects. It is worth noting that many tsetse flies carry asymptomatic infection with GpSGHV and even dissections cannot detect symptoms. In such cases the degenerate primers will greatly assist in the detection of different virus strains even if in these asymptomatic flies. An important objective of our research is the development of this virus as insect birth control. This has been hampered by the age maturation resistance exhibited by adult flies. Current research has identified that this is largely due to the formation of the peritrophic membrane. Ongoing research is keying on means to breach this barrier using various chemical and molecular technologies. In addition to developing improved formulations we have developed methods to mass infect lab reared adult flies. This spring we will be testing the ability to introduce MdSGHV into virus free fly populations to evaluate its ability to persist and spread in natural populations. Prior tests in field cages have demonstrated that introduced virus can persist in populations over at least 10 generations.
Publications
- Prompiboon, P., V.-U. Lietze, J.S.S. Denton, C.J. Geden, T. Steenberg, and D.G. Boucias. 2010. The Musca domestica salivary gland hypertrophy virus: An insect virus that globally infects and sterilizes female house flies. Applied and Environmental Microbiology 76: 994-998.
- Abd-Alla, A., D.G. Boucias, and M. Bergoin. 2010. Hytrosaviridae. Pp. 101-119 in S. Asgari and K. Johnson (Eds.), Insect Virology. Horizon Scientific Press and Caister Academic Press, Norwich, United Kingdom.
- Abd-Alla, A., T. Salem, A.G. Parker, Y. Wang, J.A. Jehle, M.J.B. Vreysen, and D. Boucias. 2010. Universal primers for rapid detection of hytrosaviruses. Journal of Virological Methods 171:280-283.
- Lietze, V.-U., T.Z. Salem, P. Prompiboon, and D.G. Boucias. 2010. Tissue tropism of the Musca domestica salivary gland hypertrophy virus. Virus Research 155:20-27.
- Lietze, V-U., A. M. M. Abd-Alla, Vreysen, M. J.B., Geden, C. J., and D.G. Boucias. 2011. Salivary gland hypertrophy viruses (SGHVs): a novel group of insect pathogenic viruses. Ann. Rev. Entomol. 56:63-80. Geden, C.J., T. Steenberg, V. Lietze, and D. Boucias. 2010. Salivary gland hypertrophy virus of house flies in Denmark: Prevalence, host range, and comparison with a Florida isolate. Journal of Vector Ecology (in press).
- Lietze, U-V., A. M. M. Abd-Alla, and D. G. Boucias 2011. Two hytrosaviruses, MdSGHV and GpSGHV, induce distinct cytopathologies in their respective host insects. J Invertebrate Pathology (in press).
- Geden, C., A. Garcia-Maruniak, V.-U. Lietze, J. Maruniak, and D. G. Boucias 2011. Impact of house fly salivary gland hypertrophy virus (MdSGHV) on a heterologous host, stable fly (Stomoxys calcitrans). J. Medical Entomology (in press).
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Progress 08/01/09 to 07/31/10
Outputs
OUTPUTS: MdSGHV has been detected in house fly samples from North America, Europe, Asia, the Caribbean, and the Southwestern Pacific. Nucleotide sequences from 16 different geographic isolates were highly homologous, and detected polymorphism was correlated with geographic source. With all isolates, 24h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. Regardless of the MdSGHV isolate tested, all susceptible insects displayed similar degrees of SGH and a complete suppression of oogenesis. House flies (Musca domestica L.) infected with MdSGHV were found in fly populations collected from 12 out of 18 Danish livestock farms. Infection rates ranged from 0.5 to 5 and averaged 1.2 percent overall. None of the stable flies, rat-tail maggot flies or yellow dung flies that were collected from virus-positive farms displayed SGH. In laboratory transmission tests, no SGH symptoms were observed in stable flies, flesh flies, black dump flies, or face flies that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands and ovaries of these species resulted in SGHV infection of the challenged house flies. The tissue tropism of Musca domestica salivary gland hypertrophy virus (MdSGHV) infecting adult house flies was examined by transmission electron microscopy (TEM) and quantitative real-time PCR. The midgut, ovary, abdominal fat body, crop, air sac and brain showed the presence of enveloped virions in vacuoles of associated tracheal cells. No sites of viral morphogenesis were detected in the tracheal cells. Analysis of MdSGHV DNA and transcript titers revealed that viral DNA and viral transcripts was present in all tissue samples collected from MdSGHV-infected flies; the highest levels in salivary glands and at the lowest levels in hemolymph. However, over the range of different tissues that were analyzed, there was no correlation between estimated quantities of genome copies and viral transcripts. The function of viral transcripts in host tissues that do not show sites of viral morphogenesis remains to be elucidated. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from e66-ODV homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47C. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The MdSGHV has been defined as a unique virus that is globally distributed. Work done on this virus and on those viruses infecting the tsetse fly has led to the description of a new virus family Hytrosaviridae that contains the genera Glossinavirus and Muscavirus. An important objective of our research is the development of this virus as insect birth control. This has been hampered by the age maturation resistance exhibited by adult flies. Current research has identified that this is largely due to the formation of the peritrophic membrane. Ongoing research is keying on means to breach this barrier using various chemical and molecular technologies. During this past year we have developed an EST data base on infected/healthy flies that has provided a framework to address the host/virus interactions. The recognition that this virus replicates in non salivary gland tissues is important in elucidating the mechanism underlying the virus induced sterility. Ongoing research is addressign the impact of this virus on the corpus cardica and JH biosynthesis. The development of universal primers for SGHVs provides a tool for detecting hytrosavirus in tsetse flies and house flies. The availability of these primers will facilitate detection of hytrosavirus in a wide range of insects and is expected to result in the discovery of other hytroviruses in other insects. It is worth noting that many tsetse flies carry asymptomatic infection with GpSGHV and even dissections cannot detect symptoms. In such cases the degenerate primers will greatly assist in the detection of different virus strains even if in these asymptomatic flies.
Publications
- Prompiboon, P., V.-U. Lietze, J.S.S. Denton, C.J. Geden, T. Steenberg, and D.G. Boucias. 2010. The Musca domestica salivary gland hypertrophy virus: An insect virus that globally infects and sterilizes female house flies. Applied and Environmental Microbiology 76: 994-998.
- Abd-Alla, A., D.G. Boucias, and M. Bergoin. 2010. Hytrosaviridae. Pp. 101-119 in S. Asgari and K. Johnson (Eds.), Insect Virology. Horizon Scientific Press and Caister Academic Press, Norwich, United Kingdom.
- Abd-Alla, A., T. Salem, A.G. Parker, Y. Wang, J.A. Jehle, M.J.B. Vreysen, and D. Boucias. 2010. Universal primers for rapid detection of hytrosaviruses. Journal of Virological Methods(doi:10.1016/j.jviromet.2010.09.025).
- Lietze,V.-U., T.Z. Salem, P. Prompiboon, and D.G. Boucias. 2010. Tissue tropism of the Musca domestica salivary gland hypertrophy virus. Virus Research (in press).
- Lietze, V-U., A. M. M. Abd-Alla, Vreysen, M. J.B., Geden, C. J., and D.G. Boucias. 2011. Salivary gland hypertrophy viruses (SGHVs): a novel group of insect pathogenic viruses. Ann. Rev.. Entomol. (in press).
- Geden, C.J., T. Steenberg, V. Lietze, and D. Boucias. 2010. Salivary gland hypertrophy virus of house flies in Denmark: Prevalence, host range, and comparison with a Florida isolate. Journal of Vector Ecology (in press).
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: The genome size and gene content, tissue tropism beyond the salivary gland, virus transmission mode and virulence characteristics of MdSGHV has been compared to the GpSGHV. In addition, the general genome organization of SGHVs has been compared to other circular dsDNA insect viruses and to obtain phylogenetic relationships. The molecular and biological data has been compiled to describe the taxonomic position of this unique virus group. One of the hallmark events of infection by SGHVs is the shutdown of oogenesis. Initially, we speculated that that the virus may be producing metabolites that interfere with egg production. However, assays involving the injection infected gland homogenates failed to suppress egg development in healthy flies suggesting that the hypertrophied gland is not producing a readily detectable inhibitory factor. Research did reveal that transcription of egg proteins in the fat body tissue of infected flies was down-regulated. This could be due to the effect of the virus on the fat body, follicular epithelium (site of ecdysone synthesis), or on the neuroendrocrine system. Amending houseflies with exogenous ecdysone stimulated transcription of the egg protein genes. Furthermore, hemolymph samples harvested at various stages post infection contained reduced levels of juvenile hormones suggesting that the virus may be infecting or disrupting the function of the corpora allata or midgut endocrine cells. The qPCR assays did show high levels of viral DNA and transcripts associated with dissected brains. A series of experiments were conducted to quantify the levels and assess the infectivity of MdSGHV released by individual infected flies via salivation and excretion. Our experimental approach was to orally challenge individual, newly emerged healthy flies with filter-sterilized homogenates of viremic salivary glands or crops, with saliva collected from individual infected flies during one feeding event, or with excreta collected from individual viremic flies over night. In addition, crops were examined by transmission electron microscopy, and saliva and excreta samples were subjected to quantitative real-time PCR analysis to estimate the viral load in these organs. We initiated a program to expand the current collection of MdSGHVs. One goal of this research was to examine the distribution and biological activity of the MdSGHV; until recently, the only research on MdSGHV has been conducted in our laboratory on a strain isolated in 2005 from a dairy in North Florida. The house fly, a cosmopolitan and synanthropic insect, can be readily collected on all continents in areas of human activity. Protocols have been established to improve the detection of virus in field collected flies; in cohorts (one thousand flies) a single infected fly can be readily detected and amplified for analysis. Secondly protocols have been established for the mass production of viremic flies suitable for introducing virus into populations lacking this biocontrol agent. Large-scale tent studies have been conducted to examine the transmission and intergenerational persistence of this virus. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
On the basis of the available morphological, (patho)biological, genome and phylogenetic data, we have proposed that the MDSGHV and GpSGHV are members of a new virus family named Hytrosaviridae. This family currently comprises two unassigned species GpSGHVand MdSGHV and tentative species MeSGHV. Here, we present the characteristics and the justification for establishing this new virus family. Direct sequencing of the 3'-RACE products revealed a total of 78 poly (A) transcripts representing 95 ORFs from the 108 putative ORFs identified in silico. Sixteen of these transcripts contained multiple putative ORFs (TCMPOs). Temporal transcription of one of the TCMPOs was observed; at one day post-infection (dpi), the monocistronic-MdSGHV084 transcript was detected, and at two dpi, the TCMPO 84/85 transcript was identified. The transcript MdSGHV045 showed splicing in its 3'-UTR with a 262 bp fragment of MdSGHV043, an ORF located 847 bases downstream. Analysis of 77 3'-UTRs indicated extensive heterogeneity in the MdSGHV polyadenylation signals and cleavage sites. In addition, twenty cis-natural antisense transcripts were found. Finally, our approach of sequencing the 3'-RACE products directly, without cloning, demonstrated that this method is a useful approach for high-throughput detection of UTRs and transcript variants of large genome DNA viruses. Oral treatments of newly emerged individual flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Infection rates did not differ between females and males or between challenged flies that were kept individually and in groups. Using quantitative real-time PCR, MdSGHV DNA was quantified in salivary secretions and excreta obtained from viremic flies. Beginning two days post-infection (dpi), saliva samples collected from individual flies during one feeding event contained detectable numbers of MdSGHV copies. Viral copy numbers increased exponentially until 4 dpi, and from 5-21 dpi each infected fly released an average 1.0 x 106 MdSGHV copies per feeding event. Oral transmission experiments showed that the virus released via salivary secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection rate. Evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that salivation onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies. MdSGHV has been detected in house fly samples from North America, Europe, Asia, the Caribbean, and the Southwestern Pacific. Results showed that infectivity varied among the different isolates but was not correlated with viral genome copy number per infected gland pair equivalent as estimated by quantitative PCR. In all cases, 24-h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. Nucleotide sequences from 16 different geographic isolates showed polymorphism that correlated with geographic source. The variation observed in biological activity does not correspond to genotypic variation.
Publications
- Lietze,V-U., Sims, K, Salem, TZ,. Geden, CJ, and. Boucias, DG. 2009. Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), via salivary secretions and excreta. J. Invertebr Pathol. 101:49-55.
- Garcia-Maruniak, A., Abd-Alla, A. M. M., Salem, T. Z., Parker, A.G., Lietze,V., van Oers, M. M., Maruniak, J .E., Kim, W.,. Burand, J.P, Cousserans F., Robinson, A. S., Vlak J. M., Bergoin, M and. Boucias, D. G. 2009. Comparative analysis of two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica. J. Gen.Virol 90: 334-346.
- Salem, T. Z., A. Garcia-Maruniak, V.-U. Lietze, J. E. Maruniak, and D. G. Boucias. 2009 Analysis of transcripts from predicted ORFs of the Musca domestica salivary gland hypertrophy virus (MdSGHV). J. General Virology 90:1270-1280.
- Abd-Alla, A. M. M., J. M. Vlak, M. Bergoin, J. E. Maruniak, A. Parker, J. P. Burand, J. A. Jehle,. and D. G. Boucias 2009. Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family. Archives of Virology 154:909-918.
- Prompiboon,P., Lietze, V.-U., Denton,J.S.S., Geden,C.J., Steenberg, T., and Boucias, D.G. 2009. The Musca domestica salivary gland hypertrophy virus: An insect virus that globally infects and sterilizes female house flies. Appl/Environ. Microbiol. (pending)
- Lietze, V. 2011. Biology of the Salivary Gland Hypertrophy Viruses. Annual Review of Entomology Volume 56 (pending).
- Lietze, V.-H., Salem, T. Z., Prompiboon,P.,and Boucias, D.G. 2009. Tissue tropism of the Musca domestica salivary gland hypertrophy virus. J. Invertebr. Pathol. (pending).
- Boucias, D.G.,Prompiboon,P.,and Lietze, V.-U., 2009. Mechanisms underlying the resistance of Musca domestica to the hytrosavirus infection. J. Gen. Virol.(pending).
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: The genome of the virus that causes salivary gland hypertrophy in Musca domestica was sequenced. Direct sequencing of the MdSGHV 3'-RACE products validated the majority of putative transcripts and provided insight into the complexity of the transcription termination of this large dsDNA animal virus. Sixteen transcripts contained multiple putative ORFs (TCMPOs) and a total of twenty cis-natural antisense transcripts (cis-NATs) were detected. The transcript, MdSGHV045, showed splicing in the 3'-UTR region. Direct sequencing of the 3'-RACE products detected transcripts that possessed variant 3'-UTRs; many transcripts contained multiple polyadenylation signals (PS) and cleavage sites (CS). Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped morphology, and possessing a large circular dsDNA genome. Both SGHVs induce similar disease symptoms, but have distinct biological, structural and molecular characteristics. MdSGHV measures 65 by 550 nm and contains a 124,279 bp genome (~44% G+C) that codes for 108 putative open reading frames (ORFs). GpSGHV contains a 190,032 bp genome (28% G+C) with 160 putative ORFs. Comparative analysis shows that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs. Overall, the amino acid identity values ranged from 19 to 39% among ORFs. The genome co-linearity of the two viral genomes was found to be significant and stronger than to the nudiviruses. Transmission ofMdSGHV within feral populations of M. domestica is mediated orally via deposition and consumption of saliva, a composite of salivary gland secretions and crop contents. Transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima as well as on the hemocoel side in close vicinity to muscle cells. No replication sites were detected in these tissues. Oral treatments of newly emerged individual flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. MdSGHV DNA was quantified in salivary secretions and excreta obtained from viremic flies. Beginning two dpi, saliva samples collected from individual flies during one feeding event contained detectable numbers of MdSGHV copies. Viral copy numbers increased exponentially until 4 dpi, and from 5-21 dpi each infected fly released an average 1.0 x 106 copies per feeding event. Oral transmission experiments showed that the virus released via salivary secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection rate. Excreta samples collected over night at 5 dpi from individual infected flies contained an average 6.5 x 105 copies per sample. Low infection rates (2%) were produced when newly emerged flies were challenged with these samples by oral treatments or injection, respectively. In house flies salivation onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies. PARTICIPANTS: Individuals: Tamer Salem (postdoc 1 month salary), Alejandra Garcia-Maruniak (senior biologist 3 months salary), Verena Lietze (senior biologist 3 months salary) Pannipa Prompiboon (post doc 3 months salary), James Maruniak (coPI) Drion Boucias (PI) Billy Kiperstock (undergraduate intern 4 months salary) Niisi Torto(undergraduate intern 4 months salary) Department of Entomology and Nematology, PO Box 110620, University of Florida, Gainesville, Florida 32611-0620, USA Collaborators and Partner Organizations: A. M. M. Abd-Alla and A. Parker Entomology Unit, FAO/IAEA Agriculture & Biotechnology Laboratory, IAEA Laboratories Seibersdorf, A-2444, Seibersdorf, Austria. Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, 1600 SW 23rd Drive, Gainesville, FL 32608, USA (Christopher Geden, James Becnel) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Noteworthy, our approach of sequencing the 3'-RACE products directly without cloning demonstrated that this method is a useful approach for high-throughput detection of UTRs and transcript variants, which terminate in close proximity, of large genome DNA viruses. Phylogenetic analysis of conserved genes clustered SGHVs separate from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV present differences, their pathology, host range and genome composition indicate they are related. To date salivary gland hypertrophy viruses (SGHVs) have been isolated from different dipteran species, such as the tse-tse fly Glossina pallidipes (GpSGHV), the house fly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). Significantly we have collected MdSGHV isolates from worldwide sources. Bioassays have shown that the different genotypes display a spectrum of biological activities. All of the SGHVs share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 550-1000 nm in length and 50-65 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120-190 kbp and having G+C ratios ranging from 28-44%; (iii) they cause overt salivary gland hypertrophy symptoms in dipteran adults leading to partial or complete sterility. Available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with other large invertebrate DNA viruses. Significantly, the great majority of SGHV ORFs could not be assigned by sequence comparison. On the basis of the available morphological, biological, genome and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This family comprises two genera Glossinavirus and Muscavirus and the unassigned species MdSGHV. Here, we present the characteristics and the justification for establishing this new virus family.
Publications
- Garcia-Maruniak, A., Maruniak; J.E., Farmerie; W. , and Boucias, D. G 2008. Sequence analysis of a non-classified, non-occluded DNA virus that causes salivary gland hypertrophy of Musca domestica, MdSGHV. Virology 377:184-196.
- Geden, CJ., Lietze, V., and Boucias, DG. 2008. Seasonal prevalence and transmission of salivary gland hyperplasia virus of house flies, Musca domestica L. (Diptera:Muscidae). J. Med. Entomol. 45(1): 42-51.
- Garcia-Maruniak ,A., Abd-Alla, A. M. M., Salem, T. Z., Parker, A.G., Lietze,V., van Oers, M. M., Maruniak, J .E., Kim, W.,. Burand, J.P, Cousserans F., Robinson, A. S., Vlak J. M., Bergoin, M and. Boucias, D. G. 2008. Comparative analysis of two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica. J. Gen.Virol (in press).
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