Genetic Characterization of Lumpy Skin Disease Viruses Circulating in Lesotho Cattle.

Lumpy skin disease is one of the fast-spreading viral diseases of cattle and buffalo that can potentially cause severe economic impact. Lesotho experienced LSD for the first time in 1947 and episodes of outbreaks occurred throughout the decades. In this study, eighteen specimens were collected from LSD-clinically diseased cattle between 2020 and 2022 from Mafeteng, Leribe, Maseru, Berea, and Mohales' Hoek districts of Lesotho. A total of 11 DNA samples were analyzed by PCR and sequencing of the extracellular enveloped virus (EEV) glycoprotein, G-protein-coupled chemokine receptor (GPCR), 30 kDa RNA polymerase subunit (RPO30), and B22R genes. All nucleotide sequences of the above-mentioned genes confirmed that the PCR amplicons of clinical samples are truly LSDV, as they were identical to respective LSDV isolates on the NCBI GenBank. Two of the elevem samples were further characterized by whole-genome sequencing. The analysis, based on both CaPV marker genes and complete genome sequences, revealed that the LSDV isolates from Lesotho cluster with the NW-like LSDVs, which includes the commonly circulating LSDV field isolates from Africa, the Middle East, the Balkans, Turkey, and Eastern Europe.

Molecular characterization of recombinant LSDV isolates from 2022 outbreak in Indonesia through phylogenetic networks and whole-genome SNP-based analysis.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and water buffaloes caused by the LSD virus, leading to high morbidity, low mortality, and a significant economic impact. Initially endemic to Africa only, LSD has spread to the Middle East, Europe, and Asia in the past decade. The most effective control strategy for LSD is the vaccination of cattle with live-attenuated LSDV vaccines. Consequently, the emergence of two groups of LSDV strains in Asian countries, one closely related to the ancient Kenyan LSDV isolates and the second made of recombinant viruses with a backbone of Neethling-vaccine and field isolates, emphasized the need for constant molecular surveillance. This current study investigated the first outbreak of LSD in Indonesia in 2022. Molecular characterization of the isolate circulating in the country based on selected LSDV-marker genes: RPO30, GPCR, EEV glycoprotein gene, and B22R, as well as whole genome analysis using several analytical tools, indicated the Indonesia LSDV isolate as a recombinant of LSDV_Neethling_vaccine_LW_1959 and LSDV_NI-2490. The analysis clustered the Indonesia_LSDV with the previously reported LSDV recombinants circulating in East and Southeast Asia, but different from the recombinant viruses in Russia and the field isolates in South-Asian countries. Additionally, this study has demonstrated alternative accurate ways of LSDV whole genome analysis and clustering of isolates, including the recombinants, instead of whole-genome phylogenetic tree analysis. These data will strengthen our understanding of the pathogens' origin, the extent of their spread, and determination of suitable control measures required.

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

A non-toxic recombinant Clostridium septicum α toxin induces protective immunity in mice and rabbits.

Clostridium septicum alpha toxin (CSA) plays significant roles in ruminant's braxy. Genetically engineered CSA has been shown to function as a potential vaccine candidate in the prevention of the disease caused by Clostridium septicum. In the present study, we synthesized a non-toxic recombinant, rCSAm4/TMD by introducing four amino acid substitutions (C86L/N296A/H301A/W342A) and 11-amino-acid deletion (residues 212 to 222). Compared to recombinant CSA, rCSAm4/TMD showed no cytotoxicity to MDCK cells and was not fatal to mice. Moreover, rCSAm4/TMD could protect immunized mice against 5 × mouse LD100 (100% lethal dose) of crude CSA without obvious pathological change. Most importantly, rabbits immunized with rCSAm4/TMD produced high titers of neutralizing antibodies which protected the rabbits against crude CSA challenge. These data suggest that genetically detoxified rCSAm4/TMD is a potential subunit vaccine candidate against braxy.

Phylogenetic analysis of Newcastle disease virus detected in Eritrea between 2017 and 2021.

Thirty-five samples collected from chickens in 13 commercial farms in Eritrea between 2017 and 2021 following reports of disease were screened for Newcastle disease virus. Seventeen samples (50%) were shown to be positive by RT-PCR. An initial analysis of partial fusion (F) gene sequences of 10 representative samples indicated that the viruses belonged to subgenotype VII.1.1. Subsequently, full F gene sequence analysis of four of these representative samples confirmed the genotype of the viruses but also revealed that they were not identical to each other suggesting different origins of the VII.1.1 subgenotype viruses circulating in Eritrea. These data have implications for the control of Newcastle disease within the poultry population in Eritrea.

Poxvirus Infections in Dairy Farms and Transhumance Cattle Herds in Nigeria.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria.

Avian influenza H5N1 in a great white pelican (Pelecanus onocrotalus), Mauritania 2022.

In February 2022, mortalities among great white pelicans (Pelecanus onocrotalus) were reported in the Parc National de Diawling, southwestern Mauritania. Samples were collected and processed, indicating the presence of high pathogenicity avian influenza subtype H5N1. A nearly complete genome was generated for one sample, revealing a high similarity [> 99.5% (H5) nucleotide sequence identity] with Clade 2.3.4.4b H5N1 identified in Europe in 2022.

Highly pathogenic avian influenza H5N1 virus outbreak among Cape cormorants (Phalacrocorax capensis) in Namibia, 2022.

In January 2022, significant mortality was observed among Cape cormorants (Phalacrocorax capensis) on the west coast of Namibia. Samples collected were shown to be positive for H5N1 avian influenza by multiplex RT-qPCR. Full genome analysis and phylogenetic analysis identified the viruses as belonging to clade 2.3.4.4b and that it clustered with similar viruses identified in Lesotho and Botswana in 2021. This is the first genomic characterization of H5N1 viruses in Namibia and has important implications for poultry disease management and wildlife conservation in the region.

First Report of Lumpy Skin Disease in Myanmar and Molecular Analysis of the Field Virus Isolates.

Lumpy skin disease virus (LSDV) causes lumpy skin disease in cattle and buffaloes, which is associated with significant animal production and economic losses. Since the 2000s, LSDV has spread from Africa to several countries in the Middle East; Europe; and Asia; including, more recently, several south-east Asian countries. In November 2020, Myanmar reported its first LSD outbreak. This study reports on the first incursion of LSD in Myanmar and the molecular analysis of the LSDV detected. Staff from the Livestock Breeding and Veterinary Department (LBVD) of the Ministry of Agriculture, Livestock, and Irrigation collected samples from cattle with suspected LSD infection. The Food and Agriculture Organization (FAO) of the United Nations' Emergency Centre for Transboundary Animal Diseases (ECTAD) and the Joint International Atomic Energy Agency (IAEA)/FAO program's Animal Health and Production laboratory provided LSDV diagnostic support to two regional veterinary diagnostic laboratories in Myanmar. Samples from 13 cattle tested positive by real-time PCR. Selected samples underwent sequence analysis in IAEA laboratories. The results show that the Myanmar LSDV sequences clustered with LSDV isolates from Bangladesh and India, LSDV Kenya, and LSDV NI-2490. Further characterization showed that the Myanmar LSDV is 100% identical to isolates from Bangladesh and India, implying a common source of introduction. These findings inform diagnosis and development of control strategies.

Molecular Characterization of the 2020 Outbreak of Lumpy Skin Disease in Nepal.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and buffaloes transmitted by blood-feeding vectors and causes high morbidity and low-to-moderate mortality. Since the first observation of LSD in Zambia in 1929, it has spread in cattle populations across African countries, the Middle East, Europe, and Asia. Following the recent outbreaks of LSD in South Asian countries such as India and Bangladesh, the disease was first reported in cattle farms in Nepal in June 2020. This study investigated the Nepalese LSD outbreak and confirmed that the disease spread rapidly to three neighboring districts in a month, infecting 1300 animals. Both cattle and buffaloes showed common clinical signs of LSD, with the exception that the buffaloes presented small nodular lesions without centered ulcerations. The collected samples were first tested for the presence of LSDV by real-time PCR. We further applied molecular tools, RPO30, GPCR, EEV glycoprotein gene, and B22R, for additional characterization of the LSDV isolates circulating in Nepal. Using a PCR-based Snapback assay, we confirmed that samples collected from cattle and buffaloes were positive of LSDV. Furthermore, sequence analysis (phylogenetic and multiple sequence alignments) of four selected LSDV genes revealed that the Nepal LSDVs resemble the Bangladesh and Indian isolates and the historic isolates from Kenya. We also highlight the importance of a unique B22R gene region harboring single-nucleotide insertions in LSDV Neethling and LSDV KSGPO-240 vaccine strains, enabling us to differentiate them from the Nepalese isolates and other fields isolates. This study demonstrates the importance of disease surveillance and the need to determine the source of the disease introduction, the extent of spread, modes of transmission, and the necessary control measures.

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.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Enhancing vector refractoriness to trypanosome infection: achievements, challenges and perspectives.

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013-2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP's major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.

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.

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control. RESULTS: In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species. CONCLUSIONS: The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

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.

Expression Profile of Glossina pallidipes MicroRNAs During Symptomatic and Asymptomatic Infection With Glossina pallidipes Salivary Gland Hypertrophy Virus (Hytrosavirus).

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) infects tsetse flies predominantly asymptomatically and occasionally symptomatically. Symptomatic infections are characterized by overt salivary gland hypertrophy (SGH) in mass reared tsetse flies, which causes reproductive dysfunctions and colony collapse, thus hindering tsetse control via sterile insect technique (SIT). Asymptomatic infections have no apparent cost to the fly's fitness. Here, small RNAs were sequenced and profiles in asymptomatically and symptomatically infected G. pallidipes flies determined. Thirty-eight host-encoded microRNAs (miRNAs) were present in both the asymptomatic and symptomatic fly profiles, while nine host miRNAs were expressed specifically in asymptomatic flies versus 10 in symptomatic flies. Of the shared 38 miRNAs, 15 were differentially expressed when comparing asymptomatic with symptomatic flies. The most up-regulated host miRNAs in symptomatic flies was predicted to target immune-related mRNAs of the host. Six GpSGHV-encoded miRNAs were identified, of which five of them were only in symptomatic flies. These virus-encoded miRNAs may not only target host immune genes but may also participate in viral immune evasion. This evidence of differential host miRNA profile in Glossina in symptomatic flies advances our understanding of the GpSGHV-Glossina interactions and provides potential new avenues, for instance by utilization of particular miRNA inhibitors or mimics to better manage GpSGHV infections in tsetse mass-rearing facilities, a prerequisite for successful SIT implementation.

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.

Comparative Analysis of Salivary Gland Proteomes of Two Glossina Species that Exhibit Differential Hytrosavirus Pathologies.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions.