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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.

Culicoides biting midges (Diptera: Ceratopogonidae) are a successful group of small (1-3 mm) haematophagous flies, some species of which are biological vectors of veterinary arboviruses, such as bluetongue virus, epizootic haemorrhagic disease virus, African horse sickness virus and Simbu serogroup viruses. In this study, we examine seasonal and spatial effects on the presence and distribution of Culicoides communities associated with ruminant and equine farms in Israel, and their infection with Simbu serogroup viruses. Our results demonstrate that both the vectors and the viruses are widely spread in Israel, including regions that were previously considered Culicoides-free. Moreover, our results show that although seasonality affects infection with Simbu serogroup viruses, both viruses and potential vectors can be found year round, suggesting continuous circulation of Simbu serogroup viruses in Israeli livestock farms. Finally, this study provides novel and basic information on Simbu serogroup-infected Culicoides in Israel: it demonstrates that Sathuperi, Shuni and Peaton viruses were circulating in Israel in 2015-2017 as they were found in C. imicola and C. oxystoma, both potential vectors of these viruses, and supplies the first-ever genomic detection of Sathuperi in Israel. Consequently, the data emerging from this study are of importance in understanding the epidemiology of arboviruses in Israel and are of relevance to the potential spread and possible future outbreaks of different Simbu serogroup viruses within the Mediterranean region and Europe.

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.

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.

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.

Viruses of the Simbu serogroup are arboviruses that are known to cause outbreaks of abortion, stillbirth and congenitally deformed neonates. This study presents the results of antibody screening of Simbu serogroup viruses in heifers born in Israel after October 2013, and in adult milking cows born before May 2012. Thirteen dairy cattle farms in five regions, and one sheep flock, entered this study. Serum samples that were found to be positive by ELISA were further tested by specific virus- neutralization test against a panel of Simbu serogroup viruses including Akabane, Aino, Sathuperi, Shamonda, and Peaton viruses. Antibody detection in lactating adult cows revealed that several viruses were circulating in Israel between 2008-2014. Moreover, during autumn 2014 the heifers became serum-positive after being exposed to more than one Simbu serogroup virus concurrently. The results of this study shed new light on Simbu virus infections in Israel, and may contribute to the epidemiology of the Simbu serogroup around the Mediterranean Basin in general.

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.

Viruses of the Simbu serogroup cause lesions to foetuses that are seen at birth and that correlate with the stage of pregnancy at which the dam first contracts the virus. The Simbu serogroup comprises arboviruses known to cause outbreaks of abnormal parturitions in domestic ruminants; these abnormalities include abortion, stillbirth, and congenitally deformed neonates. Simbu serogroup members include: Akabane virus (AKAV), Aino virus, Cache Valley virus, and Schmallenberg virus. Lately, dairy herds calf malformations have been observed in Europe, where there have been reports of clinical manifestations such as diarrhoea, fever, and reduced milk yield in adult lactating cows. The Israeli dairy cattle industry has experienced 2 major episodes of abnormal parturitions that resulted from 2 arboviral Simbu serogroup episodes, which occurred 35 years apart. A wave of apparently newly introduced AKAV was noted from the beginning of January 2012. Investigations carried out throughout the period of late Summer 2011 to early Winter 2012, associated the Israeli AKAV strain with central nervous system manifestations in lactating cows. A lack of clinical/epidemiological 'uniformity' among the AKAV infections was noted during these investigations. Here we describe and discuss the clinical and spatial distribution differences found among the 3 above-mentioned outbreaks. Comparable features in the clinical presentation, spatial distribution, and target‑animal issues relating to Akabane disease are discussed.

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.