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Important lessons have been learned by the Israeli veterinary community regarding Simbu serogroup viruses infections. This serogroup of viruses might cause the births of neonatal malformation in susceptible ruminant’s populations. Until 2012, only Akabane virus was connected with the births of malformed ruminants in Israel. However, serological and genomic detection tests, coupled with viral isolations, revealed that more than a single Simbu serogroup serotype could be present concurrently in the same farm or even in the same animal. From 2012 to date, Aino, Shuni, Shamunda, Satuperi, Peaton, Schmallenberg, and Sango viruses have been found in Israel either by serological or genomic investigation. Israel is located in the Eastern Mediterranean Basin, a terrestrial and climatic bridge between the three old continents. The Eastern Mediterranean shores benefit from both the tropical/subtropical and the continental climatic conditions. Therefore, the Eastern Mediterranean basin might serve as an optimal investigatory compound for several arboviral diseases, acting as a sentinel. This review summarizes updated information related to the presence of Simbu serogroup viruses in Israel.

Simbu serogroup are arbo‐ viruses which are mainly transmitted by Culicoides. Two members of the Simbu serogroup, Akabane and Shuni viruses, have been isolated from congenitally malformed ruminants in Israel. A recent serosurvey revealed that Israeli ruminants have been exposed to several additional Simbu viruses, including Shamonda and Sathuperi that seems to be circulating in Israel. In April 2017, an apparently healthy one‐month‐old male calf was transferred to the Kimron Veterinary Institute. A few days later, the calf was reported to be slow to respond to its surroundings and was not able to feed on its own. Blindness was observed upon clinical examination. RNA of the small, medium and large segments of Simbu serogroup viruses were amplified and sequenced from the testis tissues and from the Cerebrospinal fluid (CSF). During post‐mortem examination, hydranencephaly was defined. Phylogenetic analysis of all three segments of Simbu serogroup viruses showed that the sequences detected in the Israeli calf were virtually identical to Peaton virus (PEAV). PEAV was also detected in two pools of Culicoides imicola trapped at two different locations in Israel. This is the first genomic detection of PEAV outside Australia and Japan. These results are of epidemiological significance, as they demonstrate that PEAV is circulating in Israel and affects cattle. Consequently, these results are also of relevance to a potential spread of Simbu serogroup viruses into Europe.

Simbu serogroup viruses induce acute clinical diseases and abnormal courses of pregnancies in livestock. In Israel, two members of this serogroup, namely Akabane virus (AKAV) and Shuni virus (SHUV), were recently detected and, in Europe, Schmallenberg virus (SBV) poses a threat to ruminants. To address this emerging problem, a universal S-segment-based real-time RT-PCR assay (Uni-S) for the detection of Simbu serogroup viruses was established, which, additionally, enabled species identification of the detected viruses by subsequent sequencing. The newly developed probe-based PCR system enabled reliable detection of a comprehensive panel of Simbu viruses. Furthermore, several SBV isolates and German field samples were tested by the new Uni-S system in comparison to a SBV-specific real-time RT-PCR and both assays exhibited equally high levels of sensitivities. Finally, co-circulation of AKAV and SHUV in Israel was confirmed by analyzing field samples using the Uni-S assay followed by sequence analysis of the positive samples. To validate the test specificity, blood and tissue samples from animals negative for Simbu viruses, preparations of genetically related viruses and additional ruminant pathogens were examined and all were found to be negative. In conclusion, the new assay enabled sensitive and rapid universal molecular detection of Simbu viruses and is expected to serve as a valuable method for infection diagnosis, especially in regions where several Simbu serogroup members circulate.

In late 2011, Schmallenberg virus (SBV), a novel, arthropod-borne, teratogenic orthobunyavirus, emerged near the German/Dutch border and thereafter spread rapidly throughout the continent thereby causing great economic losses in European livestock. SBV mainly infects ruminants and closely related viruses such as Sabo virus (SABOV), Simbu virus (SIMBUV) and Sathuperi virus (SATV) have been isolated from their insect-vectors or putative ruminant hosts. However, information about their pathogenesis and in vivo studies with SABOV, SIMBUV, and SATV are scarce. As experimental infections of ruminants are comprehensive and time-consuming, an SBV small animal model was assessed regarding its suitability for studying Simbu viruses. Adult type I interferon deficient mice (IFNAR-/-) were subcutaneously infected with the Simbu serogroup members SABOV, SIMV and SATV, respectively, and compared to SBV-infected mice. All animals were clinically, virologically, serologically, and pathologically examined. The clinical signs were mainly characterised by the loss of body weight and by paralysis. In blood, and samples from the spleen and brain, high loads of viral genome were detected using newly developed real-time PCR assays. The most common histologic lesions included meningo-encephalomyelitis, perivascular cuffing of lymphocytes and macrophages, neuronal degeneration and gliosis. These lesions have also been described in foetuses after transplacental infection with SBV. In-situ hybridisation signals were widely distributed in multiple neurons of the brain and spinal cord in all examined, inoculated mice. In conclusion, IFNAR-/- mice are a suitable animal model for pathogenesis studies of a broad range of Simbu serogroup viruses since all the viruses examined displayed a common pattern of viral organ and tissue distribution in this mouse model.

Orthobunyaviruses of the Simbu serogroup are transmitted by insects (primarily biting midges) and infect mammals and/or birds. Many have been associated with disease in livestock or humans. The orthobunyavirus genome comprises three negative-sense RNA segments (L, M and S). We report the complete coding sequences of 57 isolates of Simbu serogroup viruses collected in Australia during 1968–1984. Phylogenetic analysis identified novel genogroups of Akabane virus (AKAV), Aino virus (AINOV) and Peaton virus, and provided evidence of constrained movement of AKAV between epidemiological systems in the northern and eastern regions of the continent. Differential clustering of AKAV isolates in trees inferred from L, M and S segments was indicative of intratypic segment reassortment. Similarly, intertypic segment reassortment was detected between AKAV and Tinaroo virus, and between AINOV and Douglas virus. L segments representing novel genogroups were detected in AINOV reassortants, suggesting the presence of unidentified Simbu group viruses in the episystem.

In 2015 and 2016, we observed 15 malformed calves that were exposed to intrauterine infection with Shamonda virus, a Simbu serogroup orthobunyavirus, in Japan. Characteristic manifestations were arthrogryposis and gross lesions in the central nervous system. Our results indicate that this arbovirus should be considered a teratogenic virus in ruminants.

During a study on Japanese encephalitis (JE) from Kolar district of Karnataka state, India in 1986; two virus isolates were obtained in infant Swiss albino mouse from a pig and a human serum sample. For characterization of these virus isolates, they were propagated in Vero CCL-81 cells. These virus isolates were screened for flaviviruses (Japanese encephalitis, West Nile, Dengue, Kyasanur forest disease) and Alphavirus (Chikungunya) by RT-PCR and found to be negative. Further these they were screened for bunyaviruses using genus-specific primers. A virus isolate from a human sample was sequenced using next generation sequencing; which identified it as Oya virus, Simbu group of the genus Orthobunyavirus of the family Bunyaviridae. Phylogenetic analysis of L, M, S (N and NSs) revealed its close association with Chinese strain of Oya virus in Simbu serogroup with the distance of 6.5>4.2>3.2% for nucleotides and 2.4>0.8>0.0% for the amino acid of L>M>S segments respectively. Based on the PCR results; an isolate from pig sample was also confirmed as Oya virus. This study was strengthened by findings of IgG antibody positivity against Oya virus in retrospective serum samples of suspected febrile illness cases from this area by an indigenously developed ELISA. Oya virus positivity was also recorded in human samples collected from Karnataka using nested RT-PCR. This is the first report of the presence of Oya virus in human samples. Further studies are needed to determine disease-causing potential in humans.

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatus captured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatus pools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

International audience; Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.

Schmallenberg virus (SBV) and Akabane virus (AKAV) are teratogenic Simbu serogroup Orthobunyaviruses. Embryonated chicken egg models (ECE) have been used to study the pathogenicity and teratogenicity of Simbu viruses previously, however to date no such studies have been reported for SBV. Hence, the aims of this study were to investigate if ECE are susceptible to experimental SBV infection, and to evaluate the pathogenicity and teratogenicity of SBV and AKAV in ECE models. Two studies were conducted. In Study A, SBV (106.4 TCID50) was inoculated into the yolk-sac of 6-day-old and 8-day-old ECEs. In Study B, SBV and AKAV were inoculated into 7-day-old ECEs at a range of doses (102.0–106.0 TCID50). ECE were incubated at 37 °C until day 19, when they were submitted for pathological and virological examination. SBV infection in ECE at 6, 7 and 8 days of incubation resulted in stunted growth and musculoskeletal malformations (arthrogryposis, skeletal muscle atrophy, contracted toes, distorted and twisted legs). Mortality was greater in embryos inoculated with SBV (31%) compared to AKAV (19%), (P These studies demonstrate for the first time that the ECE model is a suitable in vivo small animal model to study SBV. Furthermore, these results are consistent with the clinico-pathological findings of natural SBV and AKAV infection in ruminants.

The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, with a cosmopolitan distribution. This group of arthropod-borne viruses includes important pathogens of humans and domestic animals e.g. Oropouche orthobunyavirus and Schmallenberg virus. Sensitive and specific diagnostic tools are required for recognition and control of outbreaks. A novel TaqMan® RT-qPCR assay was developed, optimised and analytically validated for the broad detection of the Simbu serogroup orthobunyaviruses. A region in the S segment, which encodes the nucleocapsid protein, was used to design a group primer set and a pair of differently labelled TaqMan® minor groove binder probes to distinguish phylogenetic clade A and B of the serogroup. Efficiencies determined for seven members of the group were 99% for Akabane orthobunyavirus (AKAV), 96% for Simbu orthobunyavirus (SIMV), 96% for Shuni orthobunyavirus (SHUV), 97% for Sathuperi orthobunyavirus (SATV), 84% for Shamonda orthobunyavirus (SHAV), 93% for Ingwavuma virus (INGV, now classified as Manzanilla orthobunyavirus) and 110% for Sabo virus (SABOV, now classified as AKAV). The 95% limit of detection (TCID50/reaction) was 10-3.61 for AKAV, 10-2.38 for SIMV, 10-3.42 for SHUV, 10-3.32 for SATV, 10-1.67 for SHAV, 100.39 for INGV and 10-2.70 for SABOV.

The Simbu serogroup of orthobunyaviruses comprises a wide range of viruses with different medical and veterinary relevance. These viruses are known to reassort, and coinfection of the same cell is one of the prerequisites for reassortment. Here, a mammalian cell line was infected with various members of this virus group, inoculated after several time points with a second Simbu serogroup virus, and analyzed by strain or species specific immunofluorescence staining. Different virus species or different strains of the same virus species were able to co-infect mammalian cells, but only for a limited time frame. After a few hours, the replication of the first virus led to a gradual inhibition of a second virus until a complete resistance to superinfection after 24 h regardless whether it is another strain of the same virus species or a distinct member of the serogroup.

Next-generation sequencing (NGS) of agents causing idiopathic human diseases has been crucial in the identification of novel viruses. This study describes the isolation and characterization of two novel orthobunyaviruses obtained from a jungle myna and a paddy bird from Karnataka State, India. Using an NGS approach, these isolates were classified as Cat Que and Balagodu viruses belonging to the Manzanilla clade of the Simbu serogroup. Closely related viruses in the Manzanilla clade have been isolated from mosquitos, humans, birds, and pigs across a wide geographic region. Since Orthobunyaviruses exhibit high reassortment frequency and can cause acute, self-limiting febrile illness, these data suggest that human and livestock infections of the Oya/Cat Que/Manzanilla virus may be more widespread and/or under-reported than anticipated. It therefore becomes imperative to identify novel and unknown viruses in order to understand their role in human and animal pathogenesis. The current study is a step forward in this regard and would act as a prototype method for isolation, identification and detection of several other emerging viruses.

Oropouche fever has reemerged in Parauapebas and Porto de Moz municipalities, Pará State, Brazil. Serologic analysis (immunoglobulin M-ELISA) and virus isolation confirmed Oropouche virus (OROV) in both municipalities. Nucleotide sequencing of 2 OROV isolates from each location indicated genotypes I (Parauapebas) and II (Porto de Moz) in Brazil.

In 1999, two viruses were isolated from blood samples of sentinel cattle in the Western part of Japan. The physiochemical and morphological properties of these viruses indicated that they belonged to the family Bunyaviridae. Sequence analysis of the S segment indicates that the two viruses are closely related to Sathuperi virus (SATV). The N-terminal 168 amino acid of the G2 protein of the M segment was highly homologous with that of SATV (98.2%). Given these results, we conclude that the newly isolated viruses are closest to SATV, which was initially isolated in India and Nigeria over 30 years ago.

Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10 4 to 10 1 molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 10 7 OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies. The Bunyaviridae family of viruses is subdivided into five genera. Except for the plant pathogenic genus Tospovirus, the Orthobunyavirus, Hantavirus, Nairovirus, and Phlebovirus genera are composed of enzootic viruses, some of which cause zoonotic disease in humans. As is the case with many zoonoses, humans act as accidental dead-end hosts of a zoonotic transmission cycle oscillating between mammals (mostly rodents) and arthropods (Orthobunyavirus, Nairovirus, and Phlebovirus) or among rodents via aerosols and bite wounds (Hantavirus). There are 330 known viruses in these four genera, and 174 are listed as belonging to the genus Orthobunyavirus, including half of the approximately 60 Bunyaviridae viruses causing disease in humans (10, 11).

A novel virus, Schmallenberg virus (SBV), was discovered in Europe in October 2011, and since then, cases of SBV infection have been reported in sheep, cattle, and goats in several European countries (1–4). Preliminary phylogenetic analyses revealed that SBV is a member of the genus Orthobunyavirus within the family Bunyaviridae and is related to Simbu serogroup viruses (1). Similar to Akabane virus (AKAV), another Simbu serogroup virus, SBV can cause fatal congenital defects by infection of fetuses during a susceptible stage in pregnancy (2). Vaccines for SBV are not available. Thus, SBV poses a serious threat to naive populations of ruminant livestock in Europe. Orthobunyaviruses are arthropod-borne viruses with a negative-stranded tripartite RNA genome comprising large (L), medium (M), and small (S) segments. Genetic reassortment occurs naturally among these viruses, which results in the emergence of new virus strains that have altered biologic properties (5). The L segment encodes the RNA-dependent RNA polymerase; antigenic determinants are the M-encoded viral surface glycoproteins Gn and Gc, which are responsible for viral attachment, cell fusion, hemagglutination, and the induction of neutralizing antibodies, and the S-encoded nucleocapsid protein N, which plays a role in complement fixation (6). In the pregenomics era, orthobunyavirus relationships were determined solely by serologic cross-reactivity analyses (7), but since DNA sequencing became available, phylogenetic relationships have additionally been assessed by comparison of partial genome sequences (8,9). However, published full-length genome sequence information is sparse, which makes in-depth phylogenetic analysis difficult. Therefore, a detailed taxonomic classification of SBV could not be made initially when the virus emerged. The first report of SBV showed highest similarities of M- and L-segment sequences to partial Aino virus and AKAV sequences, whereas the N gene was most closely related to Shamonda virus (SHAV) (1). Additionally, results of recent investigations on complete N and M genes and partial L genes of SHAV, Douglas virus (DOUV), and Sathuperi virus (SATV) suggested that SBV is a reassortant consisting of the M segment from SATV and the S and L segments from SHAV (9). Conversely, in 2001, SHAV was described as a reassortant virus comprising the S and L segments of SATV and the M segment from the unclassified Yaba-7 virus (8). To clarify the phylogenetic relationships and classification of SBV within the Simbu serogroup, we conducted genetic and serologic investigations of its relationship to 9 other Simbu serogroup viruses.

A virus, named Oya virus, was isolated in Vero cell cultures from the lungs of a pig suspected of Nipah virus infection. The virus was revealed as a spherical enveloped RNA virus with a diameter of 79 nm. For identification of Oya virus, RT-PCR was performed. A common primer set for S-RNA of the Simbu serogroup of the genus Bunyavirus was able to amplify a cDNA from Oya virus RNA. The sequence data of the product revealed that the partial gene of Oya virus S-RNA segment had 65-70% homology with published cDNA sequences of Simbu serogroup viruses. The phylogenetic analysis of the data showed that the Oya virus is grouped in Simbu serogroup, but is genetically distinct from the serogroup viruses that have been analyzed molecularly. Serological surveys revealed that the virus distributed widely and densely in Malaysia.

Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucurui, Para state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (

NIH grants AI 43336 and AI 10984 The University of Texas Medical Branch. Departments of Microbiology and Immunology. Galveston, Texas / The University of Texas Medical Branch. Departments of Microbiology and Pathology. Galveston, Texas. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. U.S. Naval Medical Research Center. Detachment, Lima, Peru. U.S. Naval Medical Research Center. Detachment, Lima, Peru. The University of Texas Medical Branch. Departments of Microbiology and Immunology. Galveston, Texas / The University of Texas Medical Branch. Departments of Microbiology and Pathology. Galveston, Texas / The University of Texas Medical Branch. Center for Tropical Diseases. Galveston, Texas. The University of Texas Medical Branch. Departments of Microbiology and Immunology. Galveston, Texas / The University of Texas Medical Branch. Departments of Microbiology and Pathology. Galveston, Texas / The University of Texas Medical Branch. Center for Tropical Diseases. Galveston, Texas. Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.

We describe a reverse transcription-polymerase chain reaction (RT-PCR) with primers that anneal to the 5′ and 3′ ends and amplify the Bunyavirus S RNA segments. The RT-PCR was done on the fluids of C6/36 cells infected with each of 21 bunyaviruses. The bunyaviruses studied, with the exception of Catu virus, produced amplicons having 700 to 1300 base pairs and probably contained the whole S RNA segment sequence. A nested PCR performed with these amplicons distinguished California and most Bunyamwera serogroup viruses from other bunyaviruses by use of BBC specific internal primers for the S RNA segment, and distinguished Simbu serogroup viruses from others by use of BS specific internal primers. The nested-PCR amplicons of Guaroa, Maguari, California encephalitis, Bunyamwera, and Oropouche viruses were sequenced. The sequences were aligned with previously known sequences of the S RNA segment of the same viruses, showing a high degree of homology and thus confirming the specific origin of these amplicons. The nested RT-PCR is suitable as a specific screening for most California and Bunyamwera serogroup and Simbu serogroup viruses depending on the use of BBC or BS internal primers. Oropouche virus is an important public health problem in Brazil and the nested PCR with BS primers could be used for the detection of this virus in tissue culture and mouse brain isolates as well as in clinical samples.

To the Editor: Viruses in the Simbu serogroup are arboviruses that cause abortion, stillbirth, and congenital abnormalities in domestic ruminants. Akabane virus (AKAV), Aino virus (AINV), and Schmallenberg virus are the most studied in this serogroup; Shuni, Sabo, Shamonda, and Sango viruses (1,2) are examined less frequently. Until 2012, only AKAV had been associated with congenital abnormalities in Israel, although AINV had been identified serologically in dairy cow herds with no clinical signs in 2003 (3). Moreover, of 15 brain samples collected during February–October 2012 from adult cows with central nervous system manifestations, 6 were positive for AKAV by PCR. In late December 2014, the Israeli Veterinary Field Services was notified of the appearance of arthrogryposis-hydranencephaly syndrome (1) in 2 herds of sheep in the villages of Yokneam and Sde Ya’akov, respectively; both villages are located in the Izre’el Valley, in Israel’s northern valleys (Technical Appendix Figure 1), where several arboviral infections have occurred in recent decades. From our past experience (3), ≥1 virus of the Simbu serogroup was suspected to have infected the ruminants, probably during August–October 2014. We collected 27 samples of brain, placenta, spleen, lung, and blood (mixed with EDTA to prevent coagulation) from 15 sheep, goats, and cattle. Most samples were from the 2 affected flocks in the northern valley; a few were from ruminants in additional locations: Avadon, near Israel’s border with Lebanon; Ein Hachoresh, near central Israel; and Hura, close to the Negev desert (Technical Appendix Figure 1). Of the 27 samples, 23 (85%) were positive for Shuni virus (SHUV) by PCR (Table). SHUV, which had not been reported in Israel, was isolated from the brain and placenta of 1 malformed lamb (strain 2504/3/14; sample 11 in the Table). Moreover, partial nucleotide sequences of the small, medium, and large DNA segments (580/850, 4,320/4,326, and 285/6,880 bp, respectively) were identified from 3 samples (strains Yokneam 2417/2/14 and 2504/3/14 and Hura 273/14 from samples 2, 11, and 9, respectively, in the Table; Technical Appendix Figure 2). Sequence data obtained by conventional PCR in this study have been deposited into GenBank (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KP900863","term_id":"940795874","term_text":"KP900863"}}KP900863–5, {"type":"entrez-nucleotide","attrs":{"text":"KP900873","term_id":"940795878","term_text":"KP900873"}}KP900873–5, {"type":"entrez-nucleotide","attrs":{"text":"KP900879","term_id":"940795892","term_text":"KP900879"}}KP900879–80, and {"type":"entrez-nucleotide","attrs":{"text":"KP900884","term_id":"940795926","term_text":"KP900884"}}KP900884). Phylogenetic analysis of the samples showed that they were isolates of SHUV (Technical Appendix Figure 2). Additional SHUV RNA-specific fragments were detected in pathologic samples from kids, lambs, and calves (Table). Full-genome sequences were not performed, although sequencing should be done when possible to determine precise origin of isolates. Table Summary of diagnostic and laboratory findings, animal species, sample materials, and region where samples were collected in the study of Shuni virus infection in ruminants, Israel, 2014–15* For further testing, we inoculated homogenate material from 7 distinct malformations (samples 1, 2, 6, 8, 11, 12, and 15 in the Table) into baby mice; only 1 family of baby mice inoculated intracerebrally with the SHUV isolate (sample 11 in the Table) exhibited characteristic neurologic signs of nervousness. PCR confirmed that SHUV caused the cerebral infections in these mice. The isolate was also suitable for further propagation in the Vero cell line (Table). Our results showed the presence of SHUV in sheep in Israel during the winter of 2014–15 and suggest a northward expansion of SHUV from sub-Saharan Africa. Although SHUV was first isolated in the 1960s (2), its role as a pathogen has been shown only recently in its involvement in encephalitis in horses (4). We isolated SHUV from the pathologic fetal brain of a malformed lamb, an unusual laboratory finding because, although Simbu viruses are readily isolable from vectors or exposed animals during the 3 or 4 days of viremia, they are seldom isolable from pathologic specimens collected for study of congenital malformations. We deduce from the clinical evidence that malformations appear up to 6 months after infection with SHUV and after the virus has been eliminated from the host after immune activity. Thus, isolation of SHUV from malformed brains may indicate strong neurotropism of this putative pathogen. The possibility of its replication in the fetal nervous system should also be considered because an affected fetus that is born alive is likely a reservoir. Indeed, AKAV was identified in the hippocampus (only) of adult lactating cows (data not shown), and similar epidemiologic evidence might result from other Simbu virus infections. A serologic survey conducted in Israel during the 2001–2003 outbreaks of AHS showed reactivity of AINV to serum samples of ruminants in Israel’s southern regions (3). Because AINV and SHUV are known to have a strong serologic cross-reaction, SHUV has likely previously infiltrated Israel. However, whether the seroreactivity results from AINV or SHUV remains unresolved. The emergence and reemergence of arboviruses should interest medical practitioners, particularly epidemiologists. The appearance of exotic viruses in unexpected locations might result in more severe pathology in newly invaded regions than in the original arbovirus-endemic areas. Furthermore, SHUV has been detected in a child with febrile illness (2), a finding that suggests a potential zoonotic problem. Technical Appendix. Map showing locations where Shuni viruses were detected in ruminants in Israel, 2014–15, and phylogenetic trees of Simbu serogroup viruses. Click here to view.(333K, pdf)

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Seroepidemiologic studies were conducted to determine the prevalence of Oropouche (ORO) viral antibody, risk factors, and the incidence of infection among residents of the Amazon region of Peru. Blood samples, as well as demographic, cultural, and medical history data, were collected from residents in a sector of the city of Iquitos and in an adjacent rural and three neotropical rain forest communities. Blood specimens were obtained approximately one year later from a cohort of the same study subjects who were negative for ORO antibody on the initial cross-sectional survey. Sera were tested for ORO IgG antibody by an enzyme-linked immunosorbent assay. Antibody prevalences were 35% for residents of the urban population, 24-46% for the forest communities, and 18% for the rural community. Antibody prevalence increased with age, and subjects who were seropositive were significantly (P = 0.001) older (mean = 33 years) than the seronegative subjects (mean = 15 years). Multivariate analysis revealed that only age, urban and forest residence, and occupation as a farmer or housekeeper remained significantly associated with seropositivity. Seroconversion data for the same populations one year later demonstrated evidence of ORO viral infection among 28% of the residents in the rural community and 2% or less in the forest and urban communities. Oropouche virus infection was significantly associated with older age (P = 0.04) in the rural community (P < 0.001). These data support prior evidence of ORO viral infection among residents of Iquitos and surrounding villages and suggest that transmission of this virus occurs continuously in the population of this area of the Amazon basin.

The nucleoprotein genes of Akabane virus S RNA segment from 21 Japanese and two Australian isolates were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer set containing the initiation and termination codon of the gene. The RT-PCR products were sufficiently produced from the purified virion RNAs of all the isolates, and then analyzed by enzymatic digestion with 11 restriction endonucleases. Digestion with the eight restriction enzymes revealed sequence variation of the isolates. Restriction fragment length polymorphism (RFLP) profiles obtained by digestion revealed the existence of four major groups (genogroups) among the isolates. The two Australian isolates had extremely different RFLP profiles than the Japanese isolates. The data demonstrate the usefulness of analyzing the RFLP patterns to understand the genetic variability of AKA virus isolated in Japan and Australia.

In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test ( n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods’ characteristics.

Schmallenberg virus (SBV) is an emerging arbovirus infecting ruminants in Europe. SBV belongs to the Bunyaviridae family within the Simbu serogroup. Its genome comprises three segments, small (S), medium (M) and large (L), that together encode six proteins and contain NTRs. NTRs are involved in initiation and termination of transcription and in genome packaging. This study explored the 3′ mRNA termini of SBV and related Simbuviruses. In addition, the 5′ termini of SBV messenger RNA (mRNA) were characterized. For the three SBV segments, cap-snatching was found to initiate mRNA transcription both in vivo and in vitro. The presence of extraneous nucleotides between host RNA leaders and the viral termini fits with the previously described prime-and-realign theory. At the 3′ termini, common features were identified for SBV and related Simbuviruses. However, different patterns were observed for the termini of the three segments from the same virus type.

Pathogenesis of Aino virus (AIV), a suspected causative agent of congenital abnormalities of calves, has not yet been established by experimental infection of dams. To investigate the pathogenesis, 103 median tissue culture infective doses per 0.2 ml of AIV strain JaNAr 28 was inoculated into the yolk sac of 8-day-old chick embryos. At 4, 7, 10, and 13 days post-inoculation (PI) 20 eggs were opened and macro- and microscopic studies combined with virus recovery and immunohistochemical detection of the virus antigen were performed. At 7 to 13 days PI chick embryos manifested marked hydranencephaly, cerebellar hypoplasia, arthrogryposis, and scoliosis, with the highest incidences of 86.7%, 73.3%, 80,0%, and 20,0%, respectively. At 4 days PI the viral antigen was found in nerve cells, gitter cells in mild necrotic foci of the central nervous system (CNS), degenerative myotubules, and macrophages in the interstitium, which was associated with the early phase of AIV-induced encephalitis and polymyositis, with occasional accompanying hemorrhage and clumping of myotubular fragments. From 7 to 10 days PI, AIV antigen increased markedly in the liquefactive necrosis and in both degenerative and normal-looking myotubules in conjunction with developing hydranencephaly and arthrogryposis. The encephalitis and myositis had a tendency to mitigate by 10 days PI, coincident with a slight decrease in amount of AIV antigen. At 13 days PI there was almost no detectable AIV antigen in CNS and skeletal muscles, probably due to depletion of cells having affinity to AIV.

Since 2002 there has been a rise in arthrogryposis/hydranencephaly (AGH) incidence in Israel, caused by Akabane (AKA) and, possibly, Aino viruses. To test the ability to control the disease, three siRNA genes targeted to the S genome segment were designed and prepared in the form of siRNA cassettes. For the design all published S segment were aligned and two conserved target sequences with 100% homology were chosen. A third conserved target that was found exhibited only one base change found in the two Australian isolates and was also designed and tested. It was demonstrated that cells transfected with single siRNA genes showed 99% inhibition, as measured by real-time RT-PCR, virus titration and immunofluorescence. When cells were transfected with all three genes together the inhibition levels were increased and reached almost 100%.

To the Editor: In 2011, a new virus spread throughout ruminant populations in the Netherlands, Germany, and other European countries (1). Infected dairy cattle exhibited fever, reduced milk yield, loss of appetite, and diarrhea. Weeks after these signs appeared, epidemic abortions; births of malformed or stillborn animals; and perinatal deaths of calves, lambs, and goat kids were reported. Metagenomic analysis identified a novel orthobunyavirus (family Bunyaviridae), termed Schmallenberg virus (SBV), which is closely related to Akabane and Shamonda viruses (1). These arthropod-borne viruses are well-recognized ruminant pathogens in Africa, Asia, and Oceania; fetal infection is associated with an arthrogryposis–hydranencephaly syndrome. Previous reports about SBV have addressed clinical signs (2), serologic findings (3), pathologic findings (4), and SBV RNA distribution in organs of malformed calves (5) detected by real-time quantitative reverse transcription PCR (qRT-PCR). However, pathogenesis and viral cell tropism are largely unknown. Therefore, our aims were to establish an in situ hybridization method (ISH) to detect SBV mRNA, to evaluate the usefulness of this method as a complementary diagnostic tool, and to further analyze SBV pathogenesis. The in situ probe was generated according to established protocols (6). The SBV qRT-PCR product and primers used for diagnostics (5) were used to amplify an 88-bp segment of the nucleoprotein, encoded by the small segment of the SBV genome. The amplicon was cloned into a pCR4-TOPO vector (Invitrogen, Darmstadt, Germany) and sequenced. For generation of a digoxigenin-labeled antisense probe to detect viral mRNA, we used M13 reverse and SBV-S-469R primers (5). Specificity of the probe was ensured by distinct ISH signals in the brains of SBV-positive (as determined by qRT-PCR) animals (6). No reaction was detected in SBV-negative (as determined by qRT-PCR) goats, sheep, and calves; in ruminants with various non–SBV-associated nervous system lesions; or in the brain of a mouse that was experimentally infected with Akabane virus. Thereafter, we investigated SBV mRNA distribution in the central nervous system (CNS) of 82 naturally infected ruminants (46 lambs, 2 goat kids, and 34 calves), all of which had previously been found by qRT-PCR to be positive for SBV. In addition, ISH was used to examine the following tissues from 10 of these animals (4 lambs, 1 goat kid, and 5 calves): placenta, muscle, eye, heart, aorta, lung, trachea, liver, kidney, spleen, small and large intestine, mesenteric and pulmonary lymph nodes, thymus, adrenal gland, testis, and uterus. SBV mRNA was found in varying amounts, predominantly in neurons (Figure) of the cerebrum, cerebellum, brain stem, medulla oblongata, and spinal cord of 7 lambs, 1 goat kid, and 2 calves. Randomly distributed clusters of SBV-positive neurons were frequently found in small ruminants, whereas only single positive cells were found in both calves. SBV mRNA was not found in any peripheral organ. The Technical Appendix provides an overview of ISH results in relation to malformations and inflammatory CNS lesions. Histologic examination identified encephalitis, characterized by lymphohistiocytic, perivascular cuffs, in 11 (9 lambs, 1 goat kid, and 1 calf) of the 82 animals. Among animals that were positive according to ISH, all small ruminants showed inflammation, whereas both calves lacked inflammatory changes. Figure In situ detection by using Nomarski differential interference contrast microscopy of Schmallenberg virus mRNA in neurons in the medulla of a neonatal sheep for which Schmallenberg virus infection was confirmed by ... The most frequent macroscopic lesions or conditions in animals that were SBV positive by qRT-PCR, especially calves, were arthrogryposis, brachygnathia inferior, torticollis, kyphosis, lordosis, scoliosis, and muscle hypoplasia (especially in calves) (4). The predominant CNS lesions or conditions were cerebellar and cerebral hypoplasia, hydranencephaly, porencephaly, hydrocephalus, and micromyelia. Among the 8 small ruminants that were SBV positive by ISH, hydrocephalus was found in 5, cerebellar hypoplasia in 5, hydranencephaly in 1, and arthrogryposis in 6. For the 2 calves that were SBV positive, micromyelia and arthrogryposis were found in both and cerebellar hypoplasia in 1. This study indicates that neurons are the predominant target in SBV-infected neonates. Surprisingly, only 10 of 82 ruminants were positive for SBV by qRT-PCR and ISH. This discrepancy could result from low SBV mRNA copy numbers per cell, which are detectable by qRT-PCR but not ISH, and/or from reduced viral load in individual cells because of the long time between assumed infection in early gestation and time of examination after birth (≈17 weeks in sheep and 27 weeks in calves). The latter explanation is indicated by the high incidence of malformations (7) typical for teratogenic insults and might especially apply to calves. Detection of CNS inflammation in all small ruminants that were SBV positive according to ISH indicates a cause-and-effect association between high SBV mRNA copy numbers per cell and lymphohistiocytic immune response. Furthermore, low numbers of infected cells and/or reduced replication as assumed for animals that were positive by qRT-PCR but negative by ISH might explain the absence of inflammation in the calf brains. The 3 tested animals in which inflammation was found but that were SBV negative by ISH could represent an advanced resolving lesion after viral clearance or reduction. In situ detection of SBV mRNA represents a suitable way to study SBV pathogenesis, especially in the active phase of infection, and might enable identification of SBV as the causative agent in cases of CNS inflammation of unknown etiology. Technical Appendix: Absolute numbers and percentages of animals that were positive for Schmallenberg virus according to in situ hybridization in relation to central nervous system inflammation and occurrence of skeletal and brain malformations. Data refer to absolute numbers of 82 small and large ruminants that were positive for Schmallenberg virus by real-time quantitative reverse transcription PCR. Click here to view.(91K, pdf)

This report describes the first molecular characterization of Akabane virus (AKAV) in Israel. The virus was recognized by real-time RT-PCR in extracts from Culicoides imicola insects trapped at the Volcani Center located in the center of Israel. This is also the first report on the use of real-time RT-PCR to identify the virus. The quantitative capability of this technique was applied, and it was calculated that the insect extract contains 1.5 x 10(5) copies of the genome segment S. Following amplification of the small (S) genome segment, its nucleotide sequence was determined to have 93.4% identity or greater with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non-structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the non-structural protein aa sequences showed that the Israeli isolate forms a fourth cluster of AKAV, indicating a separate virus lineage. Attempts to isolate the virus by inoculation to Vero cells and by intracerebral inoculation to mice were unsuccessful.

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.

Between August and September 2000, five 2–7-year-old cows in Korea exhibited neurologic signs and were diagnosed as infected with Akabane virus based on the results of histopathology, immunohistochemistry, serology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss was noted in the brain and the ventral horn gray matter of the spinal cord. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigen was detected in the brain and spinal cord, mainly in degenerating neurons and glial cells. RTPCR analysis revealed a target band of expected size in four cows. This is the first report on an outbreak of natural Akabane virus infection in adult cattle.

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50–52%) and amino acid (30–32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743–748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

A cross-sectional serosurvey of a rural community near Iquitos, Peru was conducted to determine Oropouche (ORO) virus antibody prevalence and risk factors for human infection. Venous blood samples, and demographic, social, and risk factor data were obtained from people age five years of age and older who lived in the village of Santa Clara on the Nanay River, a tributary of the Amazon River. Sera were tested for ORO viral antibody by an ELISA. The specificity of viral antibody reactivity was determined by a standard plaque-reduction neutralization test. Interview data were analyzed by univariate and multiple logistic regression to determine which variables were statistically associated with previous ORO viral infection, as indicated by the presence of IgG antibody. Final models were evaluated based on log-likelihood and Wald chi-square. Clustering of seropositive residents within houses was analyzed by the method of Walter. Among 1,227 persons sampled, 33.7% (n=414) were positive for ORO viral IgG antibody. Overall, antibody prevalence was similar for males (33.9%) and females (33.6%), and increased significantly with age for both sexes to include more than half of persons more than 25 years of age. The length of residence in the village was positively associated with serologic status; persons who had moved to the village within the past 15 years were less likely to be seropositive than life-long residents of the same age. Antibody prevalence among immigrants who had lived in Santa Clara more than 15 years was similar to that in life-long residents. The activity most predictive of previous ORO viral infection was travel to forest communities and travel to Iquitos. No evidence of spatial heterogeneity in ORO virus antibody distribution was observed. Results suggested that endemic transmission of ORO virus in this region has been ongoing during many decades, and that people are at considerable risk of infection.

Akabane virus is a member of the genus Bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. Infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. Infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. To understand the interaction between Akabane virus and bovine brain cells, we investigated the viral tropism using primary cultures of fetal bovine brain. The cultured neuronal cells, astroglia cells and microglia cells were distinguished by cell type specific antisera. Akabane virus was found to infect neuronal cells and astroglia cells, which led to degenerative death. No microglia cells were found infected. In some brain cultures, we observed different sensitivities of the cells to two Akabane virus strains: an attenuated strain infected and spread more readily than wild type virus. This difference was not observed in a hamster fibroblast cell line. Both viral and host determinants might be involved in the different susceptibility of brain cells to Akabane virus infection.

A bovine fetus aborted at 187 days of gestation was serologically and immunohistopathologically examined. Serum and cerebrospinal fluid samples had high titers of virus-neutralizing antibody for Aino virus. A severe necrotizing encephalopathy was noted. Aino virus antigen was demonstrated in neuroglial cells within the brain lesion. The destruction of developing neuronal cells appeared to be a significant feature of the pathogenesis of lesions due to Aino virus infection in the central nervous system.

The nucleotide sequence of the small (S) RNA segment of Akabane (AKA) bunyavirus was determined. The segment is 858 nucleotides long and contains two overlapping open reading frames (ORFs), which encode the nucleocapsid (N) and nonstructural (NSs) proteins, consistent with other bunyaviruses. Comparisons with the Aino virus S RNA sequence indicated that there is 73.5% identity in nucleotide sequence. However, the sequence identity of the 5' non-coding region of the genomic RNA between these two viruses is only 55%. The N ORFs from 20 Japanese and 2 Australian isolates of AKA virus were sequenced and subjected to phylogenetic analysis. This suggested that AKA virus has evolved in multiple lineages. Twenty-three isolates were grouped into three major clusters, and the cluster which includes recent isolates was subdivided into two branches. Thus, phylogenetic analysis of the AKA virus N protein gene gives a greater insight into bunyavirus evolution.

The antigenicity and RNA genome structures of five Simbu serogroup bunyaviruses isolated in Japan and Australia were analyzed using monoclonal antibodies (Mabs) raised to Akabane (AKA) virus and oligonucleotide fingerprinting. The virion surface glycoprotein (G1) and the nucleocapsid (N) protein of heterologous viruses showed no reactivity to the Mabs, while the AKA-derived anti-G1 Mab (2F1) reacted with Peaton virus and all three AKA anti-N Mabs reacted with Tinaroo (TIN) virus at almost the same antibody titers as the homologous virus. Oligonucleotide fingerprinting analyses indicated that the three RNA species of all the viruses were unique and distinguishable. However, AKA and TIN viruses exhibited very similar S RNA oligonucleotide fingerprints, while the L and M RNA fingerprints were quite different. The S RNA sequence of TIN virus has been determined and compared with that of AKA and Aino viruses. The results revealed 95.1% S sequence homology between the AKA and TIN viruses. The antigenic and genetic comparisons of AKA and TIN viruses suggest that the two viruses may represent naturally occurring reassortant viruses.

The Bunyavirus genus, belonging to the Bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. In Australia, viruses belonging to the Simbu serogroup of the Bunyavirus genus, Akabane, Tinaroo, Peaton, Aino, Douglas, Thimiri and Facey's Paddock have been isolated. In this communication we describe two indirect ELISAs, referred to as the Simbu serogroup ELISA (SG-ELISA), and the Simbu typing ELISA (ST-ELISA), for the identification of these Simbu serogroup viruses. Infected cell lysate antigens prepared from Simbu serogroup virus isolates were assessed in the SG-ELISA for reactivity with a mouse monoclonal antibody (4H9/B11/F1). The monoclonal antibody reacted strongly with all Australian members of Simbu serogroup reference viruses and is proposed for use as a serogrouping reagent for Simbu viruses. Furthermore, the ST-ELISA enabled specific identification of viruses from within this group by recognition of characteristic reaction patterns between infected cell lysate antigens and a panel of polyclonal antisera raised to Simbu serogroup viruses.

SUMMARYSentinel herds and a vector surveillance system were used to identify the presence of arboviruses in Oman. Two strains of bluetongue virus (BTV) serotype 4 and two strains of Akabane virus, were isolated and identified. Both BTV isolates and one Akabane virus isolate came from goats while the second Akabane isolate came from Culicoides imicola. This is the first isolation of an Akabane virus from Culicoides in Arabia. Vector competence studies with the Oman viruses in laboratory reared C. variipennis showed that after oral infection both viruses replicated in Culicoides and were maintained at high titre for at least 10 days post infection.

A male fetus of gestation day 187 was aborted from a Holstein-Friesian cow in an epizootic of the Aino virus (AINOV) in September 1995. Neutralizing antibody titers against AINOV were 1:128, 1:16 and 1:64 in the dam serum, fetal ascites and cerebrospinal fluid, respectively. A 10% brain suspension of the aborted fetus was prepared immediately after autopsy, rinsed three times and sonicated before centrifugation. The supernatant was then inoculated into HmLu-1 cell cultures. A cytopathic effect was noted on post-inoculation day 7. The isolated virus was identified as the AINOV based on the physicochemical properties and cross neutralization test. This is the first report on the isolation of AINOV from an aborted bovine fetus.

The complete nucleotide sequence has been determined for the S RNA of Aino virus, a member of the Simbu serogroup ( Bunyavirus genus, family Bunyaviridae). The S RNA is 850 nucleotides long (2.76 × 10 5 dallons) and in the viral complementary sequence has a short 5' non-coding region of 34 nucleotides and a more extensive 3' non-coding region of 117 nucleotides. The 3'-5' complementarity of the Aino S RNA is about 25 residues long, depending on the arrangement. The Aino sequence predicts that, like snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses (Bishop D.H.L., et al. (1982) Nucleic Acids Res., 10, 3703–3713; Akashi H. and Bishop D.H.L. (1983) J. Virol. 45, 1155–1158), there are two S coded gene products, a nucleoprotein N, and a non-structural protein, NS S , that are read from overlapping reading frames in the viral complementary sequence. The Aino N primary gene product is composed of 233 amino acids (26.2 × 10 3 daltons) and is 45% homologous in sequence with that of LAC virus. The NS S protein of Aino virus is composed of 91 amino acids (10.5 × 10 3 daltons) and is 35% homologous in sequence with the LAC NS S protein. Unlike those viruses there are no uridylate tracts longer than 4 residues in the 5' non-coding region of the S viral RNA that could function as a template for polyadenylation of Aino S mRNA species.

The growth of Akabane virus was studied in cultures of Singh's Aedes albopictus cells, a mosquito cell line. Following a latent period of 6 to 7 hr, the virus multiplied exponentially and reached the maximum titer of about 107 PFU/ml 1 to 2 days after infection. The infection of Vero or HmLu-1 cells with Akabane virus caused marked CPE resulting in cytolysis, whereas in A. albopictus cells, no CPE was induced despite continuous release of infective virus for a long term. In A. albopictus cells, titers of extracellular virus were always shown to be 2 log-units higher than those of cell-associated virus. The virus growth in A. albopictus cell cultures was suppressed with anti-Akabane serum, and the virus produced was identified as Akabane virus by neutralization test. The infected cells were easily subcultured, and persistently infected cultures were established. Percentage of infected cells in the persistently infected cultures was very low, although progeny virus was released continuously. The virus from the persistently infected cultures seemed to retain such characteristics of the parent virus as antigenicity and pathogenicity. In plaque formation on Vero cells, however, the increase in proportion of smaller plaques was noted in the virus from the persistently infected cultures. Results obtained from the present work suggest that some species of mosquitoes might be a possible vector of Akabane virus.

Slow-reacting complement-requiring neutralizing (NT) antibody was detected in sera from cattle 2 weeks after infection with Akabane virus. Bovine sera obtained 3 or 4 weeks after infection contained slow-reacting noncomplement-requiring NT antibody. The slow-reacting complement-requiring NT antibody was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting noncomplement-requiring NT antibody was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which virus-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 60 min; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.