Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Impact of Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) on a heterologous tsetse fly host, Glossina fuscipes fuscipes.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomosis, the causal agent of sleeping sickness in humans and nagana in animals. Glossina fuscipes fuscipes is one of the most important tsetse vectors of sleeping sickness, particularly in Central Africa. Due to the development of resistance of the trypanosomes to the commonly used trypanocidal drugs and the lack of effective vaccines, vector control approaches remain the most effective strategies for sustainable management of those diseases. The Sterile Insect Technique (SIT) is an effective, environment-friendly method for the management of tsetse flies in the context of area-wide integrated pest management programs (AW-IPM). This technique relies on the mass-production of the target insect, its sterilization with ionizing radiation and the release of sterile males in the target area where they will mate with wild females and induce sterility in the native population. It has been shown that Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) infection causes a decrease in fecundity and fertility hampering the maintenance of colonies of the tsetse fly G. pallidipes. This virus has also been detected in different species of tsetse files. In this study, we evaluated the impact of GpSGHV on the performance of a colony of the heterologous host G. f. fuscipes, including the flies' productivity, mortality, survival, flight propensity and mating ability and insemination rates. RESULTS: Even though GpSGHV infection did not induce SGH symptoms, it significantly reduced all examined parameters, except adult flight propensity and insemination rate. CONCLUSION: These results emphasize the important role of GpSGHV management strategy in the maintenance of G. f. fuscipes colonies and the urgent need to implement measures to avoid virus infection, to ensure the optimal mass production of this tsetse species for use in AW-IPM programs with an SIT component.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

Prevalence of trypanosomes, salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal. RESULTS: The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected. CONCLUSION: The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

Hytrosavirus genetic diversity and eco-regional spread in Glossina species.

BACKGROUND: The management of the tsetse species Glossina pallidipes (Diptera; Glossinidae) in Africa by the sterile insect technique (SIT) has been hindered by infections of G. pallidipes production colonies with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae family). This virus can significantly decrease productivity of the G. pallidipes colonies. Here, we used three highly diverged genes and two variable number tandem repeat regions (VNTRs) of the GpSGHV genome to identify the viral haplotypes in seven Glossina species obtained from 29 African locations and determine their phylogenetic relatedness. RESULTS: GpSGHV was detected in all analysed Glossina species using PCR. The highest GpSGHV prevalence was found in G. pallidipes colonized at FAO/IAEA Insect Pest Control Laboratory (IPCL) that originated from Uganda (100%) and Tanzania (88%), and a lower prevalence in G. morsitans morsitans from Tanzania (58%) and Zimbabwe (20%). Whereas GpSGHV was detected in 25-40% of G. fuscipes fuscipes in eastern Uganda, the virus was not detected in specimens of neighboring western Kenya. Most of the identified 15 haplotypes were restricted to specific Glossina species in distinct locations. Seven haplotypes were found exclusively in G. pallidipes. The reference haplotype H1 (GpSGHV-Uga; Ugandan strain) was the most widely distributed, but was not found in G. swynnertoni GpSGHV. The 15 haplotypes clustered into three distinct phylogenetic clades, the largest contained seven haplotypes, which were detected in six Glossina species. The G. pallidipes-infecting haplotypes H10, H11 and H12 (from Kenya) clustered with H7 (from Ethiopia), which presumably corresponds to the recently sequenced GpSGHV-Eth (Ethiopian) strain. These four haplotypes diverged the most from the reference H1 (GpSGHV-Uga). Haplotypes H1, H5 and H14 formed three main genealogy hubs, potentially representing the ancestors of the 15 haplotypes. CONCLUSION: These data identify G. pallidipes as a significant driver for the generation and diversity of GpSGHV variants. This information may provide control guidance when new tsetse colonies are established and hence, for improved management of the virus in tsetse rearing facilities that maintain multiple Glossina species.

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes.

BACKGROUND: Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host's mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene). RESULTS: We show that in response to hemocoelic injections of GpSGHV into G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes. CONCLUSION: The upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography.

Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections.

Hytrosaviruses: current status and perspective.

Salivary gland hytrosaviruses (SGHVs) are entomopathogenic dsDNA, enveloped viruses that replicate in the salivary glands (SGs) of the adult dipterans, Glossina spp (GpSGHV) and Musca domestica (MdSGHV). Although belonging to the same virus family (Hytrosaviridae), SGHVs have distinct morphologies and pathobiologies. Two GpSGHV strains potentially account for the differential pathologies in lab-bred tsetse. New data suggest incorporation of host-derived cellular proteins and lipids into mature SGHVs. In addition to within the SGs, MdSGHV undergoes limited replication in the corpora allata, potentially disrupting hormone biosynthesis, and GpSGHV replicates in the milk glands providing a transmission conduit to progeny tsetse. Whereas MdSGHV is a potential biocontrol agent, the vertically transmitted GpSGHV is unsuitable for tsetse vector control but does jeopardize tsetse mass rearing.

More than one rabbit out of the hat: Radiation, transgenic and symbiont-based approaches for sustainable management of mosquito and tsetse fly populations.

Mosquitoes (Diptera: Culicidae) and tsetse flies (Diptera: Glossinidae) are bloodsucking vectors of human and animal pathogens. Mosquito-borne diseases (malaria, filariasis, dengue, zika, and chikungunya) cause severe mortality and morbidity annually, and tsetse fly-borne diseases (African trypanosomes causing sleeping sickness in humans and nagana in livestock) cost Sub-Saharan Africa an estimated US$ 4750 million annually. Current reliance on insecticides for vector control is unsustainable: due to increasing insecticide resistance and growing concerns about health and environmental impacts of chemical control there is a growing need for novel, effective and safe biologically-based methods that are more sustainable. The integration of the sterile insect technique has proven successful to manage crop pests and disease vectors, particularly tsetse flies, and is likely to prove effective against mosquito vectors, particularly once sex-separation methods are improved. Transgenic and symbiont-based approaches are in development, and more advanced in (particularly Aedes) mosquitoes than in tsetse flies; however, issues around stability, sustainability and biosecurity have to be addressed, especially when considering population replacement approaches. Regulatory issues and those relating to intellectual property and economic cost of application must also be overcome. Standardised methods to assess insect quality are required to compare and predict efficacy of the different approaches. Different combinations of these three approaches could be integrated to maximise their benefits, and all have the potential to be used in tsetse and mosquito area-wide integrated pest management programmes.

Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies.

Serotonergic Innervation of the Salivary Glands and Central Nervous System of Adult Glossina pallidipes Austen (Diptera: Glossinidae), and the Impact of the Salivary Gland Hypertrophy Virus (GpSGHV) on the Host.

Using a serotonin antibody and confocal microscopy, this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly, Glossina pallidipes Austen. Reports to date, however, note that up until this finding, dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands. Direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host, thus delaying a host response. Our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is due to a compensatory increased branching of the neurons of the salivary glands, which is associated with the increased size of the salivary glands in viral infected flies. This study shows for the first time serotonin in the cell bodies of the brain and thoracico-abdominal ganglion in adult tsetse, G. pallidipes Austen (Diptera: Glossinidae). A hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans; and, a plea is made that more research is needed to develop a blood feeding model, similar to that in the blow flies, for elucidating the various mechanisms involved in production and deployment of saliva.

Prevalence of salivary gland hypertrophy syndrome in laboratory colonies and wild flies of Glossina pallidipes in Ethiopia.

Glossina pallidipes salivary gland hyperplasia (GpSGH) syndrome caused by the salivary gland hyperplasia virus reduces the reproduction potential of tsetse flies, posing a serious threat for rearing of sufficient colonies for use of tsetse and trypanosome control using the sterile insect technique. This research was conducted in the Kaliti Tsetse Mass Rearing and Irradiation Centre in Ethiopia with the objective of studying the prevalence of GpSGH syndrome in laboratory colonies of G. pallidipes (Tororo and Arbaminch) reared for release in the implementation of the sterile insect technique and a field strain of G. pallidipes Arbaminch. Presence or absence of GpSGH was determined when pathological features of the salivary gland were revealed after dissection. The overall prevalence of GpSGH syndrome in laboratory colonies was 48.3% (747/1548) with a statistically significant (z = 17.30, p = 0.001) prevalence of 70.2% (544/775) in Arbaminch colonies and 26.26% (203/773) in Tororo colonies. The prevalence of GpSGH in laboratory flies fed according to the clean blood feeding protocol was 68.9% and 22.4% in Arbaminch and Tororo strains respectively. It was 70.5% and 27.2% respectively in laboratory colonies of Arbaminch and Tororo strains fed according to the standard membrane feeding protocol. The difference in prevalence of the disease between the two feeding protocols was not statistically significant in either Arbaminch (z = 0.361, p = 0.359) or Tororo (z = 1.22, p = 0.111) strains. The prevalence of SGH in wild G. pallidipes Arbaminch strain was 3% (15/500) and was significantly (z = 23.61, p < 0.001) lower than in the laboratory strain. The effect of age and density-related stress on the development of GpSGH was not statistically significant. The prevalence of GpSGH in the newly emerging (teneral) flies in the laboratory colonies was 66.7% and 20% in the Arbaminch and Tororo strains respectively. For all considered risk factors, the prevalence was much higher in G. pallidipes Arbaminch laboratory colonies.

Disruption of the salivary gland muscle in tsetse, Glossina pallidipes Austen, as a result of salivary gland hypertrophy virus infection.

The secretory region of the salivary glands in Glossina pallidipes Austen (Diptera: Glossinidae) is characterized by an external muscle layer. Scanning electron microscopy and transmission electron microscopy investigations provide a detailed description of the longitudinal muscle fibres and a comparison of their structure when affected by salivary gland hypertrophy virus. The virus is responsible for hypertrophy of the salivary glands in symptomatic flies, specifically of the muscle fibres, the cytoarchitecture of which is completely altered. Although observations did not reveal viral particles in the muscle cells of either asymptomatic or symptomatic flies, muscle fibres were enlarged and detached from one another and their associated basement membrane only in symptomatic flies. A decrease in type IV collagen labelling in the basement membrane of the muscles in symptomatic flies is reported and is considered a potential cause of the salivary gland muscle alteration and, possibly, myopathy. The maintenance of an organized muscular layer is essential for the normal secretion of saliva and hence its pathology in symptomatic tsetse flies could affect the normal transmission of the trypanosome that develops inside the salivary gland epithelium. Therefore, a better understanding of the possible role of the virus is essential in order to elucidate its impact on salivary deployment in symptomatic flies.

Antiviral drug valacyclovir treatment combined with a clean feeding system enhances the suppression of salivary gland hypertrophy in laboratory colonies of Glossina pallidipes.

BACKGROUND: Hytrosaviridae cause salivary gland hypertrophy (SGH) syndrome in some infected tsetse flies (Diptera: Glossinidae). Infected male and female G. pallidipes with SGH have a reduced fecundity and fertility. Due to the deleterious impact of the virus on G. pallidipes colonies, adding the antiviral drug valacyclovir to the blood diet and changing the feeding regime to a clean feeding system (each fly receives for each feeding a fresh clean blood meal) have been investigated to develop virus management strategies. Although both approaches used alone successfully reduced the virus load and the SGH prevalence in small experimental groups, considerable time was needed to obtain the desired SGH reduction and both systems were only demonstrated with colonies that had a low initial virus prevalence (SGH ≤ 10%). As problems with SGH are often only recognized once the incidence is already high, it was necessary to demonstrate that this combination would also work for high prevalence colonies. FINDINGS: Combining both methods at colony level successfully suppressed the SGH in G. pallidipes colonies that had a high initial virus prevalence (average SGH of 24%). Six months after starting the combined treatment SGH symptoms were eliminated from the treated colony, in contrast to 28 months required to obtain the same results using clean feeding alone and 21 months using antiviral drug alone. CONCLUSIONS: Combining valacyclovir treatment with the clean feeding system provides faster control of SGH in tsetse than either method alone and is effective even when the initial SGH prevalence is high.

Managing hytrosavirus infections in Glossina pallidipes colonies: feeding regime affects the prevalence of 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 10(9) virus copies in regular colonies to an average of 10(2.5) 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 10(8) virus copy numbers to an average of 10(3) 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.

Transgenerational 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 (10(9) 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.

Prevalence and genetic variation of salivary gland hypertrophy virus in wild populations of the tsetse fly Glossina pallidipes from southern and eastern Africa.

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a rod-shaped, non-occluded double-stranded DNA virus that causes salivary gland hypertrophy (SGH) and reduced fecundity in the tsetse fly G. pallidipes. High GpSGHV prevalence (up to 80%) makes it impossible to mass-rear G. pallidipes colonies for the sterile insect technique (SIT). To evaluate the feasibility of molecular-based GpSGHV management strategies, we investigated the prevalence and genetic diversity of GpSGHV in wild populations of G. pallidipes collected from ten geographical locations in eastern and southern Africa. Virus diversity was examined using a total sequence of 1497 nucleotides (≈ 1% of the GpSGHV genome) from five putative conserved ORFs, p74, pif1, pif2, pif3 and dnapol. Overall, 34.08% of the analyzed flies (n=1972) tested positive by nested PCR. GpSGHV prevalence varied from 2% to 100% from one location to another but phylogenetic and gene genealogy analyses using concatenated sequences of the five putative ORFs revealed low virus diversity. Although no correlation of the virus diversity to geographical locations was detected, the GpSGHV haplotypes could be assigned to one of two distinct clades. The reference (Tororo) haplotype was the most widely distributed, and was shared by 47 individuals in seven of the 11 locations. The Ethiopian haplotypes were restricted to one clade, and showed the highest divergence (with 14-16 single nucleotide mutation steps) from the reference haplotype. The current study suggests that the proposed molecular-based virus management strategies have a good prospect of working throughout eastern and southern Africa due to the low diversity of the GpSGHV strains.

Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus.

Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite.

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 ORFs. Here, the structural components, protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the 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 % NP-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a database search engine, a total of 45 viral proteins were identified. Of these, ten and 15 were associated with the envelope and the nucleocapsid fractions, respectively, whilst 20 were detected in both fractions, most likely representing tegument proteins. In addition, 51 host-derived proteins were identified in the proteome of the virus particle, 13 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 the development of future anti-GpSGHV strategies by interfering with virus-host interactions.

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 190,032 bp-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.