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

Global burden of 87 risk factors in 204 countries and territories, 1990�2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: Rigorous analysis of levels and trends in exposure to leading risk factors and quantification of their effect on human health are important to identify where public health is making progress and in which cases current efforts are inadequate. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 provides a standardised and comprehensive assessment of the magnitude of risk factor exposure, relative risk, and attributable burden of disease. Methods: GBD 2019 estimated attributable mortality, years of life lost (YLLs), years of life lived with disability (YLDs), and disability-adjusted life-years (DALYs) for 87 risk factors and combinations of risk factors, at the global level, regionally, and for 204 countries and territories. GBD uses a hierarchical list of risk factors so that specific risk factors (eg, sodium intake), and related aggregates (eg, diet quality), are both evaluated. This method has six analytical steps. (1) We included 560 risk�outcome pairs that met criteria for convincing or probable evidence on the basis of research studies. 12 risk�outcome pairs included in GBD 2017 no longer met inclusion criteria and 47 risk�outcome pairs for risks already included in GBD 2017 were added based on new evidence. (2) Relative risks were estimated as a function of exposure based on published systematic reviews, 81 systematic reviews done for GBD 2019, and meta-regression. (3) Levels of exposure in each age-sex-location-year included in the study were estimated based on all available data sources using spatiotemporal Gaussian process regression, DisMod-MR 2.1, a Bayesian meta-regression method, or alternative methods. (4) We determined, from published trials or cohort studies, the level of exposure associated with minimum risk, called the theoretical minimum risk exposure level. (5) Attributable deaths, YLLs, YLDs, and DALYs were computed by multiplying population attributable fractions (PAFs) by the relevant outcome quantity for each age-sex-location-year. (6) PAFs and attributable burden for combinations of risk factors were estimated taking into account mediation of different risk factors through other risk factors. Across all six analytical steps, 30 652 distinct data sources were used in the analysis. Uncertainty in each step of the analysis was propagated into the final estimates of attributable burden. Exposure levels for dichotomous, polytomous, and continuous risk factors were summarised with use of the summary exposure value to facilitate comparisons over time, across location, and across risks. Because the entire time series from 1990 to 2019 has been re-estimated with use of consistent data and methods, these results supersede previously published GBD estimates of attributable burden. Findings: The largest declines in risk exposure from 2010 to 2019 were among a set of risks that are strongly linked to social and economic development, including household air pollution; unsafe water, sanitation, and handwashing; and child growth failure. Global declines also occurred for tobacco smoking and lead exposure. The largest increases in risk exposure were for ambient particulate matter pollution, drug use, high fasting plasma glucose, and high body-mass index. In 2019, the leading Level 2 risk factor globally for attributable deaths was high systolic blood pressure, which accounted for 10·8 million (95 uncertainty interval UI 9·51�12·1) deaths (19·2% 16·9�21·3 of all deaths in 2019), followed by tobacco (smoked, second-hand, and chewing), which accounted for 8·71 million (8·12�9·31) deaths (15·4% 14·6�16·2 of all deaths in 2019). The leading Level 2 risk factor for attributable DALYs globally in 2019 was child and maternal malnutrition, which largely affects health in the youngest age groups and accounted for 295 million (253�350) DALYs (11·6% 10·3�13·1 of all global DALYs that year). The risk factor burden varied considerably in 2019 between age groups and locations. Among children aged 0�9 years, the three leading detailed risk factors for attributable DALYs were all related to malnutrition. Iron deficiency was the leading risk factor for those aged 10�24 years, alcohol use for those aged 25�49 years, and high systolic blood pressure for those aged 50�74 years and 75 years and older. Interpretation: Overall, the record for reducing exposure to harmful risks over the past three decades is poor. Success with reducing smoking and lead exposure through regulatory policy might point the way for a stronger role for public policy on other risks in addition to continued efforts to provide information on risk factor harm to the general public. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950–2019: a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2•72 (95% uncertainty interval [UI] 2•66–2•79) in 2000 to 2•31 (2•17–2•46) in 2019. Global annual livebirths increased from 134•5 million (131•5–137•8) in 2000 to a peak of 139•6 million (133•0–146•9) in 2016. Global livebirths then declined to 135•3 million (127•2–144•1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2•1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27•1% (95% UI 26•4–27•8) of global livebirths. Global life expectancy at birth increased from 67•2 years (95% UI 66•8–67•6) in 2000 to 73•5 years (72•8–74•3) in 2019. The total number of deaths increased from 50•7 million (49•5–51•9) in 2000 to 56•5 million (53•7–59•2) in 2019. Under-5 deaths declined from 9•6 million (9•1–10•3) in 2000 to 5•0 million (4•3–6•0) in 2019. Global population increased by 25•7%, from 6•2 billion (6•0–6•3) in 2000 to 7•7 billion (7•5–8•0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58•6 years (56•1–60•8) in 2000 to 63•5 years (60•8–66•1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019. Interpretation: Over the past 20 years, fertility rates have been dropping steadily and life expectancy has been increasing, with few exceptions. Much of this change follows historical patterns linking social and economic determinants, such as those captured by the GBD Socio-demographic Index, with demographic outcomes. More recently, several countries have experienced a combination of low fertility and stagnating improvement in mortality rates, pushing more populations into the late stages of the demographic transition. Tracking demographic change and the emergence of new patterns will be essential for global health monitoring. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

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