The Bunyavirales order includes at least fourteen families with diverse but related viruses, which are transmitted to vertebrate hosts by arthropod or rodent vectors. These viruses are responsible for an increasing number of outbreaks worldwide and represent a threat to public health. Infection in humans can be asymptomatic, or it may present with a range of conditions from a mild, febrile illness to severe hemorrhagic syndromes and/or neurological complications. There is a need to develop safe and effective vaccines, a process requiring better understanding of the adaptive immune responses involved during infection. This review highlights the most recent findings regarding T cell and antibody responses to the five Bunyavirales families with known human pathogens (Peribunyaviridae, Phenuiviridae, Hantaviridae, Nairoviridae, and Arenaviridae). Future studies that define and characterize mechanistic correlates of protection against Bunyavirales infections or disease will help inform the development of effective vaccines.

Iris yellow spot virus (IYSV) poses a significant threat to dry bulb onion, Allium cepa L., production and can lead to substantial yield reductions. IYSV is transmitted by onion thrips, Thrips tabaci (Lindeman), but not via seed. Transplanted onion fields have been major early season sources of IYSV epidemics. As onion thrips tend to disperse short distances, seeded onion fields bordering transplanted onion fields may be at greater risk of IYSV infection than seeded fields isolated from transplanted ones. Additionally, seeded onion fields planted early may be at greater risk of IYSV infection than those seeded later. In a 2-year study in New York, we compared IYSV incidence and onion thrips populations in seeded onion fields relative to their proximity to transplanted onion fields. In a second study, we compared IYSV incidence in onion fields with either small or large plants during midseason. Results showed similar IYSV incidence and onion thrips populations in seeded onion fields regardless of their proximity to transplanted onion fields, while IYSV incidence was over four times greater in large onion plants than in small ones during midseason. These findings suggest a greater risk of onion thrips-mediated IYSV infection in onion fields with large plants compared with small ones during midseason and that proximity of seeded fields to transplanted ones is a poor indicator of IYSV risk. Our findings on IYSV spread dynamics provided valuable insights for developing integrated pest and disease management strategies for New York onion growers.

Akabane virus (AKAV) is known as a major teratogenic agent of ruminant fetuses. In this study, we investigated the relationship between porcine abnormal deliveries and AKAV by serology, pathology, and virology investigations using specimens from 16 stillborn fetuses delivered in southern Japan between 2013 and 2015. The major clinical manifestations in stillborn fetuses were hydranencephaly, arthrogryposis, spinal curvature, and both skeletal muscle and subcutaneous edema. Histologic examination of the specimens identified atrophy of skeletal muscle fibers accompanied by adipose replacement. Nonsuppurative encephalomyelitis and decreased neuronal density in the ventral horn of the spinal cord were shown in two separate fetuses, respectively. Neutralizing antibody titers to AKAV were detected in most of the tested fetuses (13/16). The AKAV sequences detected in the affected fetuses in 2013 and 2015 were highly identical and closely related to Japanese AKAV isolates which were isolated in 2013 and sorted into genogroup I of AKAV. Immunohistochemistry visualized AKAV antigens in the neuronal cells of the central nervous system of the fetuses. These findings indicate that AKAV was involved in the birth of abnormal piglets at the affected farm. The clinical manifestations and histopathological features in the stillborn fetuses were very similar to those in ruminant neonates affected by AKAV. To avoid misdiagnosis and to evaluate the precise impact of AKAV on pig reproduction, AKAV should be considered in differential diagnoses of reproductive failures in pigs.

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

The Shuni virus (SHUV) causes an endemic viral infection in Israel and South Africa. It belongs to the Simbu serogroup within the order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus. Recently, it has been identified in aborted cases of domestic ruminants, young cattle and horses manifesting neural signs and acute death, symptomatic cows, and in carcasses of wild animals. Moreover, SHUV was isolated and identified in humans. In this study, we describe clinical cases of SHUV infection in Israeli domestic ruminants in 2020-2021, which represented clinical manifestations of simbuviral infection including abortions, a neural lethal case in a fattening calf, and an acute symptomatic case in a beef cow. In all cases, SHUV was confirmed by complete or partial viral genome sequencing. There is a significant difference of M and L segments of the novel strains compared with those of all known SHUV strains, while the S segments have more than 99% nucleotide (nt) identity with Israeli and African \"Israeli-like\" strains previously circulated in 2014-2019. This indicates a reassortment origin of the strain. At the same time, M and S segment nt sequences showed about 98-99% nt identity with some South African strains collected in 2016-2018. Nevertheless, the viral origin and the geographical place of the reassortment stayed unknown.

With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.

Proactive approaches in preventing future epidemics include pathogen discovery prior to their emergence in human and/or animal populations. Playing an important role in pathogen discovery, high-throughput sequencing (HTS) enables the characterization of microbial and viral genetic diversity within a given sample. In particular, metagenomic HTS allows the unbiased taxonomic profiling of sequences; hence, it can identify novel and highly divergent pathogens such as viruses. Newly discovered viral sequences must be further investigated using genomic characterization, molecular and serological screening, and/or in vitro and in vivo characterization. Several outbreak and surveillance studies apply unbiased generic HTS to characterize the whole genome sequences of suspected pathogens. In contrast, this study aimed to screen for novel and unexpected pathogens in previously generated HTS datasets and use this information as a starting point for the establishment of an early warning system (EWS). As a proof of concept, the EWS was applied to HTS datasets and archived samples from the 2018-9 West Nile virus (WNV) epidemic in Germany. A metagenomics read classifier detected sequences related to genome sequences of various members of Riboviria. We focused the further EWS investigation on viruses belonging to the families Peribunyaviridae and Reoviridae, under suspicion of causing co-infections in WNV-infected birds. Phylogenetic analyses revealed that the reovirus genome sequences clustered with sequences assigned to the species Umatilla virus (UMAV), whereas a new peribunyavirid, tentatively named \'Hedwig virus\' (HEDV), belonged to a putative novel genus of the family Peribunyaviridae. In follow-up studies, newly developed molecular diagnostic assays detected fourteen UMAV-positive wild birds from different German cities and eight HEDV-positive captive birds from two zoological gardens. UMAV was successfully cultivated in mosquito C6/36 cells inoculated with a blackbird liver. In conclusion, this study demonstrates the power of the applied EWS for the discovery and characterization of unexpected viruses in repurposed sequence datasets, followed by virus screening and cultivation using archived sample material. The EWS enhances the strategies for pathogen recognition before causing sporadic cases and massive outbreaks and proves to be a reliable tool for modern outbreak preparedness.

Bat flies (Hippoboscoidea: Nycteribiidae and Streblidae) are obligate hematophagous ectoparasites of bats. We collected streblid bat flies from the New World (México) and the Old World (Uganda), and used metagenomics to identify their viruses. In México, we found méjal virus (Rhabdoviridae; Vesiculovirus), Amate virus (Reoviridae: Orbivirus), and two unclassified viruses of invertebrates. Méjal virus is related to emerging zoonotic encephalitis viruses and to the agriculturally important vesicular stomatitis viruses (VSV). Amate virus and its sister taxon from a bat are most closely related to mosquito- and tick-borne orbiviruses, suggesting a previously unrecognized orbivirus transmission cycle involving bats and bat flies. In Uganda, we found mamucuso virus (Peribunyaviridae: Orthobunyavirus) and two unclassified viruses (a rhabdovirus and an invertebrate virus). Mamucuso virus is related to encephalitic viruses of mammals and to viruses from nycteribiid bat flies and louse flies, suggesting a previously unrecognized orthobunyavirus transmission cycle involving hippoboscoid insects. Bat fly virus transmission may be neither strictly vector-borne nor strictly vertical, with opportunistic feeding by bat flies occasionally leading to zoonotic transmission. Many \"bat-associated\" viruses, which are ecologically and epidemiologically associated with bats but rarely or never found in bats themselves, may actually be viruses of bat flies or other bat ectoparasites.

In 2016, the Bunyavirales order was established by the International Committee on Taxonomy of Viruses (ICTV) to incorporate the increasing number of related viruses across 13 viral families. While diverse, four of the families (Peribunyaviridae, Nairoviridae, Hantaviridae, and Phenuiviridae) contain known human pathogens and share a similar tri-segmented, negative-sense RNA genomic organization. In addition to the nucleoprotein and envelope glycoproteins encoded by the small and medium segments, respectively, many of the viruses in these families also encode for non-structural (NS) NSs and NSm proteins. The NSs of Phenuiviridae is the most extensively studied as a host interferon antagonist, functioning through a variety of mechanisms seen throughout the other three families. In addition, functions impacting cellular apoptosis, chromatin organization, and transcriptional activities, to name a few, are possessed by NSs across the families. Peribunyaviridae, Nairoviridae, and Phenuiviridae also encode an NSm, although less extensively studied than NSs, that has roles in antagonizing immune responses, promoting viral assembly and infectivity, and even maintenance of infection in host mosquito vectors. Overall, the similar and divergent roles of NS proteins of these human pathogenic Bunyavirales are of particular interest in understanding disease progression, viral pathogenesis, and developing strategies for interventions and treatments.

The Simbu serogroup of orthobunyaviruses includes several pathogens of veterinary importance, among them Schmallenberg virus (SBV), Akabane virus (AKAV) and Shuni virus (SHUV). They infect predominantly ruminants and induce severe congenital malformation. In adult animals, the intra vitam diagnostics by direct virus detection is limited to only a few days due to a short-lived viremia. For surveillance purposes the testing for specific antibodies is a superior approach. However, the serological differentiation is hampered by a considerable extent of cross-reactivity, as viruses were assigned into this serogroup based on antigenic relatedness. Here, we established a glycoprotein Gc-based triplex enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of antibodies against SBV, AKAV, and SHUV. A total of 477 negative samples of various ruminant species, 238 samples positive for SBV-antibodies, 36 positive for AKAV-antibodies and 53 SHUV antibody-positive samples were tested in comparison to neutralization tests. For the newly developed ELISA, overall diagnostic specificities of 84.56%, 94.68% and 89.39% and sensitivities of 89.08%, 69.44% and 84.91% were calculated for SBV, AKAV and SHUV, respectively, with only slight effects of serological cross-reactivity on the diagnostic specificity. Thus, this test system could be used for serological screening in suspected populations or as additional tool during outbreak investigations.