Imaging Bunyavirus Infections by Transmission Electron Microscopy: Conventional Sample Preparation vs High-Pressure Freezing and Freeze-Substitution.

Transmission electron microscopy significantly contributed to unveil the course of virus entry, replication, morphogenesis, and egress. For these studies, the most widely used approach is imaging ultrathin sections of virus-infected cells embedded in a plastic resin that is transparent to electrons. Before infiltration in a resin, cells must be processed to stabilize their components under the observation conditions in an electron microscope, such as high vacuum and irradiation with electrons. For conventional sample preparation, chemical fixation and dehydration are followed by infiltration in the resin and polymerization to produce a hard block that can be sectioned with an ultramicrotome. Another method that provides a superior preservation of cell components is high-pressure freezing (HPF) followed by freeze substitution (FS) before resin infiltration and polymerization. This chapter describes both procedures with cells infected with Bunyamwera virus (BUNV), a well characterized member of the Bunyavirales, and compares the morphological details of different viral structures imaged in the two types of samples. Advantages, disadvantages, and applications of conventional processing and HPF/FS are also presented and discussed.

Antiviral Activity of Acetylsalicylic Acid against Bunyamwera Virus in Cell Culture.

The Bunyavirales order is a large group of RNA viruses that includes important pathogens for humans, animals and plants. With high-throughput screening of clinically tested compounds we have looked for potential inhibitors of the endonuclease domain of a bunyavirus RNA polymerase. From a list of fifteen top candidates, five compounds were selected and their antiviral properties studied with Bunyamwera virus (BUNV), a prototypic bunyavirus widely used for studies about the biology of this group of viruses and to test antivirals. Four compounds (silibinin A, myricetin, L-phenylalanine and p-aminohippuric acid) showed no antiviral activity in BUNV-infected Vero cells. On the contrary, acetylsalicylic acid (ASA) efficiently inhibited BUNV infection with a half maximal inhibitory concentration (IC50) of 2.02 mM. In cell culture supernatants, ASA reduced viral titer up to three logarithmic units. A significant dose-dependent reduction of the expression levels of Gc and N viral proteins was also measured. Immunofluorescence and confocal microscopy showed that ASA protects the Golgi complex from the characteristic BUNV-induced fragmentation in Vero cells. Electron microscopy showed that ASA inhibits the assembly of Golgi-associated BUNV spherules that are the replication organelles of bunyaviruses. As a consequence, the assembly of new viral particles is also significantly reduced. Considering its availability and low cost, the potential usability of ASA to treat bunyavirus infections deserves further investigation.

Digoxin is a potent inhibitor of Bunyamwera virus infection in cell culture.

Drug repurposing is a valuable source of new antivirals because many compounds used to treat a variety of pathologies can also inhibit viral infections. In this work, we have tested the antiviral capacity of four repurposed drugs to treat Bunyamwera virus (BUNV) infection in cell cultures. BUNV is the prototype of the Bunyavirales order, a large group of RNA viruses that includes important pathogens for humans, animals and plants. Mock- and BUNV-infected Vero and HEK293T cells were treated with non-toxic concentrations of digoxin, cyclosporin A, sunitinib and chloroquine. The four drugs inhibited BUNV infection with varying potency in Vero cells, and all except sunitinib also in HEK293T cells, with digoxin rendering the lowest half maximal inhibitory concentration (IC50). Since digoxin rendered the best results, we selected this drug for a more detailed study. Digoxin is an inhibitor of the Na+/K+ ATPase, a plasma membrane enzyme responsible for the energy-dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells and involved in many signalling pathways. Digoxin was shown to act at an early time point after viral entry reducing the expression of the viral proteins Gc and N. Effects on the cell cycle caused by BUNV and digoxin were also analysed. In Vero cells, digoxin favoured the transition from G1 phase of the cell cycle to S phase, an effect that might contribute to the anti-BUNV effect of digoxin in this cell type. Transmission electron microscopy showed that digoxin impedes the assembly of the characteristic spherules that harbour the BUNV replication complexes and the morphogenesis of new viral particles. Both BUNV and digoxin induce similar changes in the morphology of mitochondria that become more electron-dense and have swollen cristae. The alterations of this essential organelle might be one of the factors responsible for digoxin-induced inhibition of viral infection. Digoxin did not inhibit BUNV infection in BHK-21 cells that have a digoxin-resistant Na+/K+ ATPase, which suggests that the effects of the blockade of this enzyme is a key factor of the antiviral activity of digoxin in BUNV-infected Vero cells.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

Evidence of circulation of Orthobunyaviruses in diverse mosquito species in Kwale County, Kenya.

BACKGROUND: Arbovirus surveillance and recurrence of outbreaks in Kenya continues to reveal the re-emergence of viruses of public health importance. This calls for sustained efforts in early detection and characterization of these agents to avert future potential outbreaks. METHODS: A larval survey was carried out in three different sites in Kwale County, Vanga, Jego and Lunga Lunga. All containers in every accessible household and compound were sampled for immature mosquitoes. In addition, adult mosquitoes were also sampled using CO2-baited CDC light traps and BG-Sentinel traps in the three sites and also in Tsuini. The mosquitoes were knocked down using trimethylamine and stored in a liquid nitrogen shipper for transportation to the laboratory where they were identified to species, pooled and homogenized ready for testing. RESULTS: A total of 366 houses and 1730 containers were inspected. The House Index (HI), Container Index (CI) and Breateau Index (BI) for Vanga Island were (3%: 0.66: 3.66) respectively. In Jego, a rural site, the HI, CI and BI were (2.4%: 0.48: 2.4) respectively. In Lunga Lunga, a site in an urban area, the HI, CI and BI were (22.03%: 3.97: 29.7) respectively. The indices suggest that this region is at risk of arbovirus transmission given they were above the WHO threshold (CI > 1, HI > 1% and BI > 5). The most productive containers were the concrete tanks (44.4%), plastic tank (22.2%), claypot (13.3%), plastic drums (8.9%), plastic basins (4%), jerricans (1.2%) and buckets (0.3%). Over 20,200 adult mosquitoes were collected using CDC light traps, and over 9,200 using BG- sentinel traps. These mosquitoes were screened for viruses by inoculating in Vero cells. Eleven Orthobunyavirus isolates were obtained from pools of Ae. pembaensis (4), Ae. tricholabis (1), Cx. quinquefasciatus (3), Culex spp. (1) and Cx. zombaensis (2). Five of the Orthobunyaviruses were sequenced and four of these were determined to be Bunyamwera viruses while one isolate was found to be Nyando virus. One isolate remained unidentified. CONCLUSIONS: These results indicate circulation of Orthobunyaviruses known to cause diverse grades of febrile illness with rash in humans in this region and highlights the need for continued monitoring and surveillance to avert outbreaks.

Association of Batai Virus Infection and Encephalitis in Harbor Seals, Germany, 2016.

We isolated Batai virus from the brain of a euthanized, 26-year-old, captive harbor seal with meningoencephalomyelitis in Germany. We provide evidence that this orthobunyavirus can naturally infect the central nervous system of a mammal. The full-genome sequence showed differences from a previously reported virus isolate from a mosquito in Germany.

A Review of Bunyamwera, Batai, and Ngari Viruses: Understudied Orthobunyaviruses With Potential One Health Implications.

Bunyamwera (BUNV), Batai (BATV), and Ngari (NRIV) are mosquito-borne viruses of the Bunyamwera serogroup in the Orthobunyavirus genus of the Bunyaviridae family. These three viruses have been found to cause disease in both livestock animals, avian species, and humans. Thus, these viruses pose a potential threat to human public health, animal health, and food security. This is especially the case in the developing nations, where BUNV and NRIV are found, mainly in Africa. BUNV and BATV are fairly well characterized, while NRIV is not well characterized owing to only sporadic detection in human and animal populations in Africa. Reassortment is common among bunyaviruses, but NRIV is believed to be the only natural reassortant of the Bunyamwera serogroup. It resulted from a combination of BUNV S and L segments and the BATV M segment. This indicates at least some level co-circulation of BUNV and BATV, which have no historically been reported to overlap in geographic distributions. But as these viruses are undercharacterized, there remains a gap in the understanding of how such reassortment could occur, and the consequences of such. Due to their combined wide range of hosts and vectors, geographic distributions, potential severity of associated diseases, and potential for transmissibility between vertebrate hosts, these viruses represent a significant gap in knowledge with important One Health implications. The goal of this review is to report available knowledge of and identify potential future directions for study of these viruses. As these are collectively understudied viruses, there is a relative paucity of data; however, we use available studies to discuss different perspectives in an effort to promote a better understanding of these three viruses and the public and One Health threat(s) they may pose.

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase.

The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.

First isolation of Bunyamwera virus (Bunyaviridae family) from horses with neurological disease and an abortion in Argentina.

Bunyamwera virus (BUNV) is the prototype virus for both the Orthobunyavirus genus and the Bunyaviridae family. Different strains of BUNV have been associated with clinical diseases in domestic animals, mainly ruminants. During 2013, in Argentina's Santa Fe Province, three new isolates of BUNV were recovered from the brain and spleen of two horses with encephalitis, and from the brain of an aborted equine fetus. This isolation of BUNV from domestic animals provided the first association of BUNV infection with disease of the central nervous system and abortion in equines in Argentina.

Detection of Orthobunyavirus in mosquitoes collected in Argentina.

Bunyamwera virus (BUNV) (Bunyaviridae, genus Orthobunyavirus, serogroup Bunyamwera) is considered an emerging pathogen for humans and animals in American countries. The CbaAr-426 strain of BUNV was recovered from mosquitoes Ochlerotatus albifasciatus (Diptera: Culicidae) collected in Córdoba province (Argentina), where serological studies detected high seroprevalences in humans and animals. Molecular detection of Orthobunyavirus was performed in mosquitoes collected in Córdoba province. Seventeen mosquito pools of Oc. albifasciatus, Ochlerotatus scapularis and Culex quinquefasciatus (Diptera: Culicidae) showed positive results; four of these positive pools, all of Oc. scapularis, were sequenced. All amplicons grouped with BUNV in the Bunyamwera serogroup. The findings highlight the circulation of BUNV in Córdoba province and represent the first report of BUNV-infected Oc. scapularis mosquitoes in Argentina.

Vector Competence of Selected Mosquito Species in Kenya for Ngari and Bunyamwera Viruses.

Bunyamwera and Ngari viruses have been isolated from a range of mosquito species in Kenya but their actual role in the maintenance and transmission of these viruses in nature remains unclear. Identification of the mosquito species efficient in transmitting these viruses is critical for estimating the risk of human exposure and understanding the transmission and maintenance mechanism. We determined the vector competence of, Aedes aegypti (L.), Culex quinquefasciatus Say, and Anopheles gambiae Giles for transmission of Bunyamwera and Ngari viruses. Ae. aegypti was moderately susceptible to Bunyamwera virus infection at days 7 and 14. Over 60% of Ae. aegypti with a midgut infection developed a disseminated infection at both time points. Approximately 20% more mosquitoes developed a disseminated infection at day 14 compared with day 7. However, while Ae. aegypti was incompetent for Ngari virus, An. gambiae was moderately susceptible to both viruses with dissemination rates more than double by day 14. Cx. quinquefasciatus was refractory to both Bunyamwera and Ngari viruses. Our results underscore the need to continually monitor emergent arboviral genotypes circulating within particular regions as well as vectors mediating these transmissions to preempt and prevent their adverse effects. The genetic mechanism for species specificity and vector competence owing to reassortment needs further investigation.

The variability of the large genomic segment of Tahyna orthobunyavirus and an all-atom exploration of its anti-viral drug resistance.

Tahyna virus (TAHV), a member of the Bunyaviridae family (California complex), is an important but neglected human mosquito-borne pathogen. The virus genome is composed of three segments, i.e., small (S), medium (M), and large (L). Previous studies on genetic variability of viruses within the California complex were focused on S and M segments, but the L segment remains relatively unstudied. To assess the genetic variation and the relation to virus phenotype we analyzed the L segment sequences of biologically diverse TAHV strains isolated in the Czech Republic and Slovakia. Phylogenetic analysis covering all available sequences of the L segment of TAHV clearly revealed two distinguished lineages, tentatively named as "European" and "Asian". The L segment strains within the European lineage are highly conserved (identity 99.3%), whilst Asian strains are more genetically diverse (identity 97%). Based on sequence comparison with other bunyaviruses, several non-synonymous nucleotide substitutions unique for TAHV in the L segment were identified. We also identified specific residue substitutions in the endonuclease domain of TAHV compared with the La Crosse virus. Since the endonuclease domain of the La Crosse virus has been resolved, we employed an all energy landscape algorithm to analyze the ligand migration of a viral polymerase inhibitor. This allowed us to demonstrate, at the atomic level, that this viral polymerase inhibitor randomly explored the specific residue substitutions in the endonuclease domain of the TAHV L segment.

Molecular characterization of the African orthobunyavirus Ilesha virus.

Ilesha virus is an arthropod-borne virus belonging to the genus Orthobunyavirus of the Bunyaviridae family. Ilesha virus has been isolated from humans in several African countries, mostly in relation with febrile illness and erythema, though there are reported cases of fatal meningoencephalitis and hemorrhagic fever. In the present study, we report the complete genomic sequence of all three Ilesha virus segments (S, M, L) and characterize the open reading frames. The nucleoprotein encoded by segment S contains 59 conserved orthobunyavirus amino acids putatively critical for protein function. For the polyprotein encoded by segment M, potential proteolytic cleavage sites and N-glycosylation sites as well as conserved cysteines are described in reference to other orthobunyaviruses. Within the C terminal glycoprotein Gc a putative fusion peptide could be localized. In the RNA-dependent RNA polymerase encoded by segment L, all strictly conserved amino acids within the four conserved regions known to be catalytically active are present. Phylogenetic analyses conducted for each Ilesha virus genomic segment confirm the classification of Ilesha virus within the Bunyamwera serogroup of orthobunyaviruses. Ilesha virus segments S and L exhibit highest genetic conservation with Bunyamwera virus and Ngari virus, with maximum sequence identities of 88% for segment S and 82% for segment L. However, the M segment was found to be more diverse with a maximum nucleotide identity of 72% to Bunyamwera serogroup viruses.