ICTV Virus Taxonomy Profile: Hytrosaviridae.

Hytrosaviridae is a family of large, rod-shaped, enveloped entomopathogenic viruses with dsDNA genomes of 120-190 kbp. Hytrosaviruses (also known as salivary gland hypertrophy viruses) primarily replicate in the salivary glands of adult dipteran flies. Hytrosaviruses infecting the haematophagous tsetse fly and the filth-feeding housefly are assigned to two genera, Glossinavirus and Muscavirus, respectively. Whereas muscavirus infections are only overt, glossinavirus infections can be either covert or overt. Overt infections are characterized by diagnostic salivary gland hypertrophy and cause either partial or complete infertility. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hytrosaviridae, which is available at ictv.global/report/hytrosaviridae.

Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization.

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.

Diversity of large DNA viruses of invertebrates.

In this review we provide an overview of the diversity of large DNA viruses known to be pathogenic for invertebrates. We present their taxonomical classification and describe the evolutionary relationships among various groups of invertebrate-infecting viruses. We also indicate the relationships of the invertebrate viruses to viruses infecting mammals or other vertebrates. The shared characteristics of the viruses within the various families are described, including the structure of the virus particle, genome properties, and gene expression strategies. Finally, we explain the transmission and mode of infection of the most important viruses in these families and indicate, which orders of invertebrates are susceptible to these pathogens.

Reprint of: Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.

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.

Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.

Expression strategy of Aedes albopictus densovirus.

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.

Papilio polyxenes densovirus has an iteravirus-like genome organization.

The genome of Papilio polyxenes densovirus was cloned and sequenced and contained 5,053 nucleotides (nt), including inverted terminal repeats (ITRs) of 271 nt with terminal hairpins of 175 nt. Its DNA sequence and monosense organization with 3 open reading frames (ORFs) are typical of the genus Iteravirus in the subfamily Densovirinae of the Parvoviridae.

Iteravirus-like genome organization of a densovirus from Sibine fusca Stoll.

The complete genome of Sibine fusca densovirus was cloned and sequenced. The genome contained 5,012 nucleotides (nt), including inverted terminal repeats (ITRs) of 230 nt with terminal hairpins of 161 nt. Its DNA sequence and monosense organization with 3 open reading frames (ORFs) is typical of the genus Iteravirus in the subfamily Densovirinae of the Parvoviridae.

Organization of the ambisense genome of the Helicoverpa armigera Densovirus.

A natural densovirus (DNV) of a serious phytophagous pest, Helicoverpa armigera, was isolated. The genome of HaDNV contained 6,039 nucleotides (nt) and included inverted terminal repeats (ITRs) of 545 nt with terminal Y-shaped hairpins of 126 nt. Its DNA sequence and ambisense organization with four typical open reading frames (ORFs) demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.

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

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

The Melolontha melolontha entomopoxvirus fusolin protein is a chitin-active lytic polysaccharide monooxygenase that displays extreme stability.

Spindles are intracellular crystals of the fusolin protein that enhances the oral virulence of insect poxviruses by disruption of the larval chitinous peritrophic matrix. The enigmatic fusolin protein is classified as a lytic polysaccharide monooxygenase (LPMO) by sequence and structure. Although circumstantial evidence points towards a role for fusolin in chitin degradation, no biochemical data exist to verify this claim. In the present study, we demonstrate that fusolin released from over 40-year-old spindles, stored for 10 years at 4 °C, are chitin-degrading LPMOs. Not only was fusolin active after long-term storage, but it also withstood high temperature and oxidative stress in its crystalline form, highlighting extreme stability that is beneficial to viral persistence and desirable for potential biotechnology applications.

The Acheta domesticus densovirus, isolated from the European house cricket, has evolved an expression strategy unique among parvoviruses.

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses.

Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome.

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts.

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 genomes of viruses isolated from Glossina pallidipes (GpSGHV) and Musca domestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programmes 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 and 50 kDa, respectively, were identified by Western analysis using a 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 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 protein of GpSGHV, detected by using immunogold-electron microscopy. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic option.

Structure and expression strategy of the genome of Culex pipiens densovirus, a mosquito densovirus with an ambisense organization.

The genome of all densoviruses (DNVs) so far isolated from mosquitoes or mosquito cell lines consists of a 4-kb single-stranded DNA molecule with a monosense organization (genus Brevidensovirus, subfamily Densovirinae). We previously reported the isolation of a Culex pipiens DNV (CpDNV) that differs significantly from brevidensoviruses by (i) having a approximately 6-kb genome, (ii) lacking sequence homology, and (iii) lacking antigenic cross-reactivity with Brevidensovirus capsid polypeptides. We report here the sequence organization and transcription map of this virus. The cloned genome of CpDNV is 5,759 nucleotides (nt) long, and it possesses an inverted terminal repeat (ITR) of 285 nt and an ambisense organization of its genes. The nonstructural (NS) proteins NS-1, NS-2, and NS-3 are located in the 5' half of one strand and are organized into five open reading frames (ORFs) due to the split of both NS-1 and NS-2 into two ORFs. The ORF encoding capsid polypeptides is located in the 5' half of the complementary strand. The expression of NS proteins is controlled by two promoters, P7 and P17, driving the transcription of a 2.4-kb mRNA encoding NS-3 and of a 1.8-kb mRNA encoding NS-1 and NS-2, respectively. The two NS mRNAs species are spliced off a 53-nt sequence. Capsid proteins are translated from an unspliced 2.3-kb mRNA driven by the P88 promoter. CpDNV thus appears as a new type of mosquito DNV, and based on the overall organization and expression modalities of its genome, it may represent the prototype of a new genus of DNV.

Genome analysis of a Glossina pallidipes salivary gland hypertrophy virus reveals a novel, large, double-stranded circular DNA virus.

Several species of tsetse flies can be infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). Infection causes salivary gland hypertrophy and also significantly reduces the fecundity of the infected flies. To better understand the molecular basis underlying the pathogenesis of this unusual virus, we sequenced and analyzed its genome. The GpSGHV genome is a double-stranded circular DNA molecule of 190,032 bp containing 160 nonoverlapping open reading frames (ORFs), which are distributed equally on both strands with a gene density of one per 1.2 kb. It has a high A+T content of 72%. About 3% of the GpSGHV genome is composed of 15 sequence repeats, distributed throughout the genome. Although sharing the same morphological features (enveloped rod-shaped nucleocapsid) as baculoviruses, nudiviruses, and nimaviruses, analysis of its genome revealed that GpSGHV differs significantly from these viruses at the level of its genes. Sequence comparisons indicated that only 23% of GpSGHV genes displayed moderate homologies to genes from other invertebrate viruses, principally baculoviruses and entomopoxviruses. Most strikingly, the GpSGHV genome encodes homologues to the four baculoviral per os infectivity factors (p74 [pif-0], pif-1, pif-2, and pif-3). The DNA polymerase encoded by GpSGHV is of type B and appears to be phylogenetically distant from all DNA polymerases encoded by large double-stranded DNA viruses. The majority of the remaining ORFs could not be assigned by sequence comparison. Furthermore, no homologues to DNA-dependent RNA polymerase subunits were detected. Taken together, these data indicate that GpSGHV is the prototype member of a novel group of insect viruses.

Quantitative PCR analysis of the salivary gland hypertrophy virus (GpSGHV) in a laboratory colony of Glossina pallidipes.

Many species of tsetse flies can be infected by a virus that causes salivary gland hypertrophy (SGH) and virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies having 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 symptomatic flies in relation to virus copy number, a quantitative PCR (qPCR) method was developed. The qPCR analyses revealed that in asymptomatic flies virus copy number averaged 1.68E+5, 2.05E+5 and 1.07E+7log(10) in DNA from an excised leg, salivary glands and a whole fly, respectively. In symptomatic flies the virus copy number in the same organs averaged 1.34E+7, 1.42E+10 and 1.5E+9, respectively. Despite these statistically significant differences (p<<0.0001) in virus copy number between asymptomatic and symptomatic flies, there was no correlation between age and virus copy number for either sets in adult flies. A clear correlation between virus copy number in pupae and their mothers was observed. Reverse transcription quantitative PCR (RT-qPCR) of the viral messenger RNA encoding ODV-E66, an envelope protein, revealed a clear correlation between virus copy number and the level of gene expression with values of 2.77log(10) in asymptomatic males and 6.10log(10) in symptomatic males. Taken together these results confirm the close relationship between virus copy number and SGH syndrome. They demonstrate the vertical transmission of GpSGHV from mother to progeny, and suggest that the development of SGH may be correlated to the virus copy number acquired by the larva during its intra-uterine development.

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.

Development of a non-destructive PCR method for detection of the salivary gland hypertrophy virus (SGHV) in tsetse flies.

A PCR based diagnostic method to detect salivary gland hypertrophy virus (SGHV) in tsetse flies is described. Two sets of primers GpSGHV1F/GpSGHV1R and GpSGHV2F/GpSGHV2R were selected from a virus-specific sequence. Both primer sets can detect specifically the virus in individual tsetse flies by generating an amplicon of 401 bp. Attempts were made to develop a simple and reliable non-destructive virus detection method in live flies. PCR reactions were performed on either crude or purified tsetse DNA from saliva and legs. While saliva can be an indicator for the presence of the virus in flies, the method is laborious. Crude extract from an excised middle leg resulted in a positive PCR reaction equivalent to crude extract from whole fly. However, sensitivity could be significantly increased when purified DNA was used as the template. In conclusion, PCR using a purified DNA template from a single tsetse leg represents an efficient, non-destructive method for virus diagnosis in live tsetse flies.