Proteomic analysis of Glossina pallidipes salivary gland hypertrophy virus virions for immune intervention in tsetse fly colonies

Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The viruses isolated from Glossina pallidipes (GpSGHV) and Musca somestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have a reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programs to control and eradicate tsetse populations in the wild. A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins. Two major GpSGHV virion proteins of about 130 kDa and 50 kDa, respectively, were identified by Western analysis using polyclonal rabbit antibody raised against whole GpSHGV virions. The proteome of GpSGHV, containing the antigens responsible for the immune-response, was investigated by liquid chromatography tandem mass spectrometry (LC-MS/MS) and 61 virion proteins were identified by comparison with the genome sequence. Specific antibodies were produced in rabbits against seven candidate proteins including the ORF10 / C-terminal fragment, ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102), PIF-2 (ORF53), PIF-3 (ORF76) and P74 (ORF1). Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope of GpSGHV using immunogold-EM. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic optio

Managing hytrosavirus infections in Glossina pallidipes colonies: Feeding regime affects the prevalence of the salivary gland hypertrophy syndrome

Many species of tsetse flies are infected by a virus that causes salivary gland hypertrophy (SGH) syndrome and the virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies with SGH have a reduced fecundity and fertility. Due to the deleterious impact of SGHV on G. pallidipes colonies, several approaches were investigated to develop a virus management strategy. Horizontal virus transmission is the major cause of the high prevalence of the GpSGHV in tsetse colonies. Implementation of a “clean feeding” regime (fresh blood offered to each set of flies so that there is only one feed per membrane), instead of the regular feeding regime (several successive feeds per membrane), was among the proposed approaches to reduce GpSGHV infections. However, due to the absence of disposable feeding equipment (feeding trays and silicone membranes), the implementation of a clean feeding approach remains economically difficult. We developed a new clean feeding approach applicable to large-scale tsetse production facilities using existing resources. The results indicate that implementing this approach is feasible and leads to a significant reduction in virus load from 109 virus copies in regular colonies to an average of 102.5 and eliminates the SGH syndrome from clean feeding colonies by28 months post implementation of this approach. The clean feeding approach also reduced the virus load from an average of 108 virus copy numbers to an average of 103 virus copies and SGH prevalence of 10% to 4% in flies fed after the clean fed colony. Taken together, these data indicate that the clean feeding approach is applicable in large-scale G. pallidipes production facilities and eliminates the deleterious effects of the virus and the SGH syndrome in these colonies

Virology, Epidemiology and Pathology of Glossina Hytrosavirus, and Its Control Prospects in Laboratory Colonies of the Tsetse Fly, Glossina pallidipes (Diptera; Glossinidae)

The Glossina hytrosavirus (family Hytrosaviridae) is a double-stranded DNA virus with rod-shaped, enveloped virions. Its 190 kbp genome encodes 160 putative open reading frames. The virus replicates in the nucleus, and acquires a fragile envelope in the cell cytoplasm. Glossina hytrosavirus was first isolated from hypertrophied salivary glands of the tsetse fly, Glossina pallidipes Austen (Diptera; Glossinidae) collected in Kenya in 1986. A certain proportion of laboratory G. pallidipes flies infected by Glossina hytrosavirus develop hypertrophied salivary glands and midgut epithelial cells, gonadal anomalies and distorted sex-ratios associated with reduced insemination rates, fecundity and lifespan. These symptoms are rare in wild tsetse populations. In East Africa, G. pallidipes is one of the most important vectors of African trypanosomosis, a debilitating zoonotic disease that afflicts 37 sub-Saharan African countries. There is a large arsenal of control tactics available to manage tsetse flies and the disease they transmit. The sterile insect technique (SIT) is a robust control tactic that has shown to be effective in eradicating tsetse populations when integrated with other control tactics in an area-wide integrated approach. The SIT requires production of sterile male flies in large production facilities. To supply sufficient numbers of sterile males for the SIT component against G. pallidipes, strategies have to be developed that enable the management of the Glossina hytrosavirus in the colonies. This review provides a historic chronology of the emergence and biogeography of Glossina hytrosavirus, and includes researches on the infectomics (defined here as the functional and structural genomics and proteomics) and pathobiology of the virus. Standard operation procedures for viral management in tsetse mass-rearing facilities are proposed and a future outlook is sketched

Trans-generational transmission of the Glossina pallidipes hytrosavirus depends on the presence of a functional symbiome

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny

Proteomic footprints of a member of Glossinavirus (Hytrosaviridae): An expeditious approach to virus control strategies in tsetse factories

The Glossinavirus (Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)) is a rod-shaped enveloped insect virus containing a 190,032bp-long, circular dsDNA genome. The virus is pathogenic for the tsetse fly Glossina pallidipes and has been associated with the collapse of selected mass-reared colonies. Maintenance of productive fly colonies is critical to tsetse and trypanosomiasis eradication in sub-Saharan Africa using the Sterile Insect Technique. Proteomics, an approach to define the expressed protein complement of a genome, was used to further our understanding of the protein composition, morphology, morphogenesis and pathology of GpSGHV. Additionally, this approach provides potential targets for novel and sustainable molecular-based antiviral strategies to control viral infections in tsetse colonies. To achieve this goal, identification of key protein partners involved in virus transmission is required. In this review, we integrate the available data on GpSGHV proteomics to assess the impact of viral infections on host metabolism and to understand the contributions of such perturbations to viral pathogenesis. The relevance of the proteome findings to tsetse and trypanosomiasis management in sub-Sahara Africa is also considered

Dynamics of the salivary gland hypertrophy virus in laboratory colonies of Glossina pallidipes (Diptera: Glossinidae)

Many species of tsetse flies are infected by a virus that causes salivary gland hypertrophy (SGH) and the virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies with SGH have a reduced fecundity and fertility. To better understand the impact of this virus in a laboratory colony of G. pallidipes, where the majority of flies are infected but asymptomatic, and to follow the development of SGH in the offspring of symptomatic infected flies, we examined the progeny of tsetse flies reared under different conditions. The results show that the progeny of asymptomatic parents did not develop SGH, while the progeny of symptomatic female flies mated with asymptomatic males developed a high rate of SGH (65% in male and 100% in females) and these flies were sterile. Stress in the form of high fly density in holding cages (180 flies/cage) and high temperature (30 °C) in the holding room did not affect the prevalence of the SGH. The virus is excreted in the saliva and there is a strong correlation between the infection status (negative, slight or strong by PCR) and the numbers of virus particles released into the blood on which the flies were fed. On average, around 102 and 107 virus particles were found in the blood after feeding asymptomatic or symptomatic infected flies respectively. Feeding the flies on new blood at every feed for three generations caused a significant reduction in the virus copy number in these flies when compared with the virus copy number in flies fed under the normal feeding regime. The results of these studies allowed the initiation of colony management protocols that aim to minimize the risk of horizontal transmission and to enable the establishment of colonies with a low virus prevalence or possibly even those that are virus fre

Correlation between structure, protein composition, morphogenesis and cytopathology of Glossina pallidipes salivary gland hypertrophy virus

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rod-shaped, enveloped virions. Its 190-kb genome contains 160 putative protein-coding open reading frames (ORFs). Here, structural components, protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: nucleocapsid, tegument, envelope, and helical surface projections, were observed in purified GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within the nuclei of infected salivary gland cells, whereas enveloped virions were located in the cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes disintegrated. Treatment of virions with 1% Nonidet P-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by 12% SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by LC-MS/MS. Using the MaxQuant program with Andromeda as a database search engine, a total of forty-five viral proteins were identified. Of these, ten and fifteen were associated with the envelope and the nucleocapsid fractions respectively, while twenty were detected in both fractions, most likely representing tegument proteins. In addition, fifty-one host-derived proteins were identified in the proteome of the virus particle, thirteen of which were verified to be incorporated into the mature virion using a proteinase K protection assay. This study provides important information about GpSGHV biology and suggests options for development of future anti-GpSGHV strategies by interfering with virus-host interactions

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500¿ 1,000 nm in length and 50¿100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The 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 baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus famil

Improving Sterile Insect Technique (SIT) for tsetse flies through research on their symbiont and pathogens

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the trypanosomes, which cause human African trypanosomosis (HAT) or sleeping sickness in humans and African animal trypanosomosis (AAT) or nagana in animals. Due to the lack of effective vaccines and inexpensive drugs for HAT, and the development of resistance of the trypanosomes against the available trypanocidal drugs, vector control remains the most efficient strategy for sustainable management of these diseases. Among the control methods used for tsetse flies, Sterile Insect Technique (SIT), in the frame of area-wide integrated pest management (AW-IPM), represents an effective tactic to suppress and/or eradicate tsetse flies. One constraint in implementing SIT is the mass production of target species. Tsetse flies harbor obligate bacterial symbionts and salivary gland hypertrophy virus which modulate the fecundity of the infected flies. In support of the future expansion of the SIT for tsetse fly control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a six year Coordinated Research Project (CRP) entitled “Improving SIT for Tsetse Flies through Research on their Symbionts and Pathogens”. The consortium focused on the prevalence and the interaction between the bacterial symbionts and the virus, the development of strategies to manage virus infections in tsetse colonies, the use of entomopathogenic fungi to control tsetse flies in combination with SIT, and the development of symbiont-based strategies to control tsetse flies and trypanosomosis. The results of the CRP and the solutions envisaged to alleviate the constraints of the mass rearing of tsetse flies for SIT are presented in this special issue