Pathogenicity and teratogenicity of Schmallenberg virus and Akabane virus in experimentally infected chicken embryos.

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 < 0.01), suggesting that SBV was more embryo-lethal. However, embryos infected with AKAV had a significantly higher prevalence of stunted growth (P < 0.05) and musculoskeletal malformations (P < 0.01), suggesting that AKAV was more teratogenic in this model. 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.

Congenital Malformations of Calves Infected with Shamonda Virus, Southern Japan.

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.

Isolation and characterization of Oya virus a member of Simbu serogroup, family Bunyaviridae, isolated from Karnataka, India.

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.

Experimental infection of suckling mice by subcutaneous inoculation with Oropouche virus.

Oropouche virus, of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, is an important causative agent of arboviral febrile illness in Brazil. An estimated 500,000 cases of Oropouche fever have occurred in Brazil in the last 30 years, with recorded cases also in Panama, Peru, Suriname and Trinidad. We have developed an experimental model of Oropouche virus infection in neonatal BALB/c mouse by subcutaneous inoculation. The vast majority of infected animals developed disease on the 5th day post infection, characterized mainly by lethargy and paralysis, progressing to death within 10 days. Viral replication was documented in brain cells by in situ hybridization, immunohistochemistry and virus titration. Multi-step immunohistochemistry indicated neurons as the main target cells of OROV infection. Histopathology revealed glial reaction and astrocyte activation in the brain and spinal cord, with neuronal apoptosis. Spleen hyperplasia and mild meningitis were also found, without viable virus detected in liver and spleen. This is the first report of an experimental mouse model of OROV infection, with severe involvement of the central nervous system, and should become useful in pathogenesis studies, as well as in preclinical testing of therapeutic interventions for this emerging pathogen.

Reemergence of Oropouche fever, northern Brazil.

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.

Jatobal virus is a reassortant containing the small RNA of Oropouche virus.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruí, Pará 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 (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

Teratogenicity of Australian Simbu serogroup and some other Bunyaviridae viruses: the embryonated chicken egg as a model.

The use of embryonated chicken eggs as a model for assessing the teratogenic potential of animal viruses was investigated with 12 members of the Bunyaviridae family. Infection of 4-day-old embryonated chicken eggs via the yolk sac with 10 of the viruses resulted in deaths or congenital deformities that were similar to those observed in Akabane virus infections of fetal ruminants and included arthrogryposis, scoliosis, mandible defects, and retarded development. Statistical analysis showed that the viruses fell into three main groupings, namely, those that caused both death and deformities (Akabane, Aino, Tinaroo, and Belmont viruses), those that mainly caused death (Peaton, Thimiri, and Facey's Paddock viruses), and those that required very high doses to cause either death or deformities (Douglas and CSIR0296 viruses). In addition, two viruses (Kowanyama and Mapputta viruses) caused neither death nor deformities. A difference in the pathogenic potential between two Akabane isolates (B8935 and CSIR016) in the embryonated chicken egg model was found to correlate with differences previously observed in experimentally infected sheep; Akabane CSIR016 was the more pathogenic. It is concluded that the embryonated chicken egg model should also be of value in assessing the teratogenic potential of other Bunyaviridae and attenuated vaccine viruses, although it does not assess the ability of the virus to cross the placenta.

An attenuated strain of Akabane virus: a candidate for live virus vaccine.

An attempt was made to attenuate the high virulent OBE-1 strain of Akabane virus by adaptation to low temperature. In it the virus was subjected to passage through HmLu-1 cell cultures at 30 degrees C. Cloning was carried out on the virus which had undergone 20 passages through these cultures to select a strain adapted to low temperature. Finally, ten clones were obtained. As a result, nine strains of clone in which virus replication was poor in HmLu-1 cell cultures at 40 degrees C were obtained. Of them, five strains of clone produced uniform plaques. Of these strains, one, or the TS-C2 strain, was selected. It was considerably lower both in peripheral infectivity to suckling mice and in intracerebral infectivity to 3-week-old mice than the OBE-1 strain. Calves and pregnant cows inoculated with the TS-C2 strain by the intracerebral, intravenous, or subcutaneous route were free from pyrexia, leukopenia, and viremia. Virus recovery was negative from various organs and fetuses. All the animals inoculated, however, were found to have neutralizing antibody produced. The results mentioned above suggested that the TS-C2 strain might have been so attenuated as to be available as a candidate strain for a live virus vaccine.

Deformities of chick embryos in experimental Akabane virus infection.

A material containing 10(3.0) approximately 10(5.0) TCID50 of Akabane virus was inoculated into 6-day-old chick embryos by the yolk sac route. Death of embryos did not increase in the course of embryonic development till 18 days of age, as compared with control groups. Later than 18 days of age, however, the numbers of dead and peeping but unhatched embryos increased, making the hatching rate significantly low. Deformities, such as arthrogryposis and hydranencephaly, appeared in almost all the dead and unhatched embryos. They were severe in dead embryos, considerably severe in peeping unhatched embryos, and comparatively mild, though highly frequent, in affected hatched chicks. Many of the hatched chicks manifested ataxia, abnormal gait, astasia, or tremor of body or legs separately or together. Virus growth was demonstrated in chick embryos inoculated at 7 days of age. The virus titer was the highest (10(3.25 approximately 10(3.75) TCID50/O.1g) in head, trunk, and muscle, and the second highest (10(2.0) approximately 10(2.5) in brain, heart, and other visceral organs. It was the highest (10(4.0) approximately 10(4.5)) in muscle and a mixture of cerebellum and brain stem in embryos inoculated at 8 days of age.