Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Characterization of experimental Shuni virus infection in the mouse.

Shuni virus (SHUV), an orthobunyavirus of the Simbu serogroup, was initially isolated in Nigeria in the 1960s, further detected in other African countries and in the Middle East, and is now endemic in Israel. Transmitted by blood-sucking insects, SHUV infection is associated with neurological disease in cattle and horses, and with abortion, stillbirth, or the birth of malformed offspring in ruminants. Surveillance studies also indicated a zoonotic potential. This study aimed to test the susceptibility of the well-characterized interferon (IFN)-α/ß receptor knock-out mouse model (Ifnar-/-), to identify target cells, and to describe the neuropathological features. Ifnar-/-mice were subcutaneously infected with two different SHUV strains, including a strain isolated from the brain of a heifer showing neurological signs. The second strain represented a natural deletion mutant exhibiting a loss of function of the S-segment-encoded nonstructural protein NSs, which counteracts the host's IFN response. Here it is shown that Ifnar-/-mice are susceptible to both SHUV strains and can develop fatal disease. Histological examination confirmed meningoencephalomyelitis in mice as described in cattle with natural and experimental infections. RNA in situ hybridization was applied using RNA Scope™ for SHUV detection. Target cells identified included neurons and astrocytes, as well as macrophages in the spleen and gut-associated lymphoid tissue. Thus, this mouse model is particularly beneficial for the evaluation of virulence determinants in the pathogenesis of SHUV infection in animals.

Differentiation of Antibodies against Selected Simbu Serogroup Viruses by a Glycoprotein Gc-Based Triplex ELISA.

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

Shuni virus-induced meningoencephalitis after experimental infection of cattle.

Shuni virus (SHUV), an insect-transmitted orthobunyavirus of the Simbu serogroup within the family Peribunyaviridae, may induce severe congenital malformations when naïve ruminants are infected during gestation. Only recently, another clinical presentation in cattle, namely neurological disease after postnatal infection, was reported. To characterize the course of the disease under experimental conditions and to confirm a causal relationship between the virus and the neurological disorders observed in the field, six calves each were experimentally inoculated (subcutaneously) with two different SHUV strains from both clinical presentations, that is encephalitis and congenital malformation, respectively. Subsequently, the animals were monitored clinically, virologically and serologically for three weeks. All animals inoculated with the 'encephalitis strain' SHUV 2162/16 developed viremia for three to four consecutive days, seroconverted, and five out of six animals showed elevated body temperature for up to three days. No further clinical signs such as neurological symptoms were observed in any of these animals. However, four out of six animals developed a non-suppurative meningoencephalitis, characterized by perivascular cuffing and glial nodule formation. Moreover, SHUV genome could be visualized in brain tissues of the infected animals by in situ hybridization. In contrast to the 'encephalitis SHUV strain', in animals subcutaneously inoculated with the strain isolated from a malformed newborn (SHUV 2504/3/14), which expressed a truncated non-structural protein NSs, a major virulence factor, no viremia or seroconversion, was observed, demonstrating an expected severe replication defect of this strain in vivo. The lack of viremia further indicates that virus variants evolving in malformed foetuses may represent attenuated artefacts as has been described for closely related viruses. As the neuropathogenicity of SHUV could be demonstrated under experimental conditions, this virus should be included in differential diagnosis for encephalitis in ruminants, and cattle represent a suitable animal model to study the pathogenesis of SHUV.

Long-term presence of tick-borne encephalitis virus in experimentally infected bank voles (Myodes glareolus).

Tick-borne encephalitis virus (TBEV) is a vector-borne pathogen that can cause serious neurological symptoms in humans. Across large parts of Eurasia TBEV is found in three traditional subtypes: the European, the Siberian and the Far-eastern subtype. Small mammalian animals play an important role in the transmission cycle as they enable the spread of TBEV among the vector tick population. To assess the impact of TBEV infection on its natural hosts, outbred bank voles (Myodes glareolus) were inoculated with one out of four European TBEV strains. Three of these TBEV strains were recently isolated in Germany. The forth one was the TBEV reference strain Neudörfl. Sampling points at 7, 14, 28, and 56 days post inoculation allowed the characterization of the course of infection. At each time point, six animals per strain were euthanized and eleven organ samples (brain, spine, lung, heart, small and large intestine, liver, spleen, kidney, bladder, sexual organ) as well as whole blood and serum samples were collected. The majority of bank voles (92/96) remained clinically unaffected after the inoculation with TBEV, but still developed a systemic infection during the first week, which transitioned to a viraemia and an infestation of the brain in some animals for the remainder of the first month. Viral RNA was found in whole blood samples of several animals (50/96), but only in a small fraction of the corresponding serum samples (4/50). From the whole blood, virus was successfully reisolated in cell culture until 14 days after inoculation. Less than five percent of all inoculated bank voles (4/96) displayed signs of distress in combination with a rapid weight loss and had to be euthanized prematurely. Overall, the recently isolated TBEV strains showed marked differences, such as a more frequent development of long-term viraemia and a higher detection rate of viral RNA in various organs, in comparison to the reference strain Neudörfl. Overall, our data suggest that the bank vole is a potential amplifying host in the TBEV transmission cycle and appears to be highly adapted to circulating TBEV strains.

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

Culicoides Biting Midges-Underestimated Vectors for Arboviruses of Public Health and Veterinary Importance.

Culicoides biting midges, small hematophagous dipterans, are the demonstrated or putative vectors of multiple arboviruses of veterinary and public health importance. Despite its relevance in disease spread, the ceratopogonid genus Culicoides is still a largely neglected group of species, predominantly because the major human-affecting arboviruses are considered to be transmitted by mosquitoes. However, when a pathogen is detected in a certain vector species, a thorough search for further vectors often remains undone and, therefore, the relevant vector species may remain unknown. Furthermore, for many hematophagous arthropods, true vector competence is often merely suspected and not experimentally proven. Therefore, we aim to illuminate the general impact of Culicoides biting midges and to summarize the knowledge about biting midge-borne disease agents using the order Bunyavirales, the largest and most diverse group of RNA viruses, as an example. When considering only viruses evidentially transmitted by Culicoides midges, the Simbu serogroup (genus Orthobunyavirus) is presumably the most important group within the virus order. Its members are of great veterinary importance, as a variety of simbuviruses, e.g., the species Akabane orthobunyavirus or Schmallenberg orthobunyavirus, induces severe congenital infections in pregnant animals. The major zoonotic representative of this serogroup occurs in South and Central America and causes the so-called Oropouche fever, an acute febrile illness in humans.