Schmallenberg virus (SBV), discovered in Europe in 2011, causes mild transient
disease in adult ruminants, but fetal infection can lead to severe
malformation in cattle, sheep and goats.To elucidate the pathogenesis of this
novel orthobunyavirus, considerable efforts are required. A reliable and
standardized infection model is essential for in vivo studies. In the present
study, two groups of four cattle were inoculated with either serum passaged in
cattle only or cell culture-grown virus. The replication of culture-grown SBV
in cattle was reduced compared to virus inoculated via infectious serum. In a
second experiment, the infectious serum was titrated in calves; the tested
batch contained 102.83 infectious doses per mL. Hence, serum-borne virus that
was only passaged in the natural host is a suitable option for a standardized
SBV infection model

Beer, Martin

Breithaupt, Angele

Eschbaumer, Michael

Hoffmann, Bernd

Wernike, Kerstin

Institutional Repository of the Freie Universität Berlin

VETERINARY RESEARCHWernike et al. Veterinary Research 2012, 43:84http://www.veterinaryresearch.org/content/43/1/84RESEARCH Open AccessSchmallenberg virus challenge models in cattle:infectious serum or culture-grown virus?Kerstin Wernike1, Michael Eschbaumer1,3, Angele Breithaupt2,4, Bernd Hoffmann1 and Martin Beer1*AbstractSchmallenberg virus (SBV), discovered in Europe in 2011, causes mild transient disease in adult ruminants, but fetalinfection can lead to severe malformation in cattle, sheep and goats.To elucidate the pathogenesis of this novel orthobunyavirus, considerable efforts are required. A reliable andstandardized infection model is essential for in vivo studies. In the present study, two groups of four cattle wereinoculated with either serum passaged in cattle only or cell culture-grown virus. The replication of culture-grownSBV in cattle was reduced compared to virus inoculated via infectious serum. In a second experiment, the infectiousserum was titrated in calves; the tested batch contained 102.83 infectious doses per mL. Hence, serum-borne virusthat was only passaged in the natural host is a suitable option for a standardized SBV infection model.IntroductionIn 2011, Schmallenberg virus (SBV), an orthobunyavirusrelated to viruses of the Simbu serogroup, was discov-ered in Germany [1,2]. In adult cattle, SBV infection isassociated with mild transient disease, but it can lead tosevere fetal malformations when pregnant cows andewes are exposed in early to mid pregnancy [3].Currently, the knowledge about SBV is very limited; areliable and highly standardized infection model isrequired to elucidate its pathogenesis and to test poten-tial vaccines.To this end, the present study compares the inductionand progression of viremia in cattle for the two differentinocula, (i) an SBV field strain that was only passaged incattle (delivered as infectious serum) as well as (ii) virusgrown in tissue culture for a maximum of 4 passages. Inaddition, the infectious serum was titrated in calves toquantify its infectivity.Materials and methodsAll experimental protocols were reviewed by a state eth-ics commission and have been approved by the compe-tent authority (State Office for Agriculture, Food Safetyand Fisheries of Mecklenburg-Vorpommern, Rostock,* Correspondence: martin.beer@fli.bund.de1Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10,Greifswald, Insel Riems 17493, GermanyFull list of author information is available at the end of the article© 2012 Wernike et al.; licensee BioMed CentraCommons Attribution License (http://creativecreproduction in any medium, provided the orGermany, ref. LALLF M-V TSD/7221.3-1.1-004/12). Allanimals were between 9 and 18 month of age.Infectious serum as inoculumIn a previous study, a single calf had been subcutane-ously inoculated with blood samples from four diseasedcattle [1] of the same outbreak series that were positivein an SBV real-time quantitative reverse transcriptionPCR (RT-qPCR). EDTA-treated whole blood, taken fromthis calf 4 days after infection (dpi) was subcutaneouslyinjected into two further calves (C01, C02). Both inocu-lated animals were PCR-positive for the first time at2 dpi, and one developed diarrhea for several days. Halfa liter of serum from each calf was collected at 3 dpi,divided into portions of 1 mL and stored at −70°C untilit was used in the subsequent experiments.The sera contained 2.0 × 103 and 1.3 × 103 50% tissueculture infectious doses per mL (TCID50/mL), respect-ively, as determined by end-point titration on baby ham-ster kidney (BHK) cells (cell line L0164, Collection ofCell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Insel Riems, Germany). Both sera were testedfree of any bacterial contamination. Using sensitive real-time PCR protocols, it could be shown that they did notcontain detectable nucleic acids of bovine herpesvirustype 1 [4], pestiviruses [5], Rift Valley fever virus [6],bluetongue virus, foot-and-mouth disease virus or epizo-otic hemorrhagic disease virus (unpublished assays).l Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.Figure 1 Real-time RT-PCR results of cattle inoculated withinfectious serum or cell culture supernatant. All cattle, eitherinoculated with infectious serum or culture-grown virus, scoredpositive in the RT-qPCR for several days. In the in-contact animalsSBV RNA was not detected at any time.Wernike et al. Veterinary Research 2012, 43:84 Page 2 of 4http://www.veterinaryresearch.org/content/43/1/84Cell culture supernatant as inoculumSBV was isolated from the blood of an infected cow aspreviously described [1]. After initial isolation on KCcells (cell line L1062; derived from Culicoides variipennismidges [7]) the virus was passaged in BHK cells, then inKC and again in BHK cells. The infectivity was deter-mined by end-point titration on BHK cells.Animal experimentsComparison between infectious serum and cell culturesupernatantTwelve SBV-naive calves were assigned to three groupsof four animals each. Calves in group 1 (C03-C06) weresubcutaneously injected with 1 mL of serum obtainedfrom C01, while calves in group 2 (C07-C10) received2 × 107 TCID50 of culture-grown virus. Animals ingroup 3 (C11-C14) were injected with phosphate-buffered saline (PBS) and kept as controls.Following the inoculation, the animals were monitoredfor the presence of clinical signs every day, rectal bodytemperatures were measured daily and serum sampleswere taken daily during the first eight days and weeklythereafter. RNA was extracted using the MagNA PureLC Total Nucleic Acid Isolation Kit for automatedextraction (Roche Diagnostics Deutschland GmbH,Mannheim, Germany) according to the manufacturer’srecommendations. SBV genome load in the samples wasdetermined by S segment-specific RT-qPCR as describedpreviously [8] with an external standard based on the Sgenome segment; at its peak, the difference between thegroups was statistically evaluated with a t-test. Sero-logical data was collected at weekly intervals using acommercially available ELISA kit (ID ScreenW Schmal-lenberg virus Indirect ELISA kit, IDvet, Montpellier,France). A diverse panel of tissue samples (e.g. lymph-nodes, spleen) was taken at necropsy on days 24 or 25;they were homogenized and tested for the presence ofSBV RNA by RT-qPCR.In vivo titration of infectious serumIn a subsequent in vivo titration experiment two calvesreceived the undiluted serum of C01 (group A, C15-C16), while groups of three calves each were givendilutions of 1/10 (group B, C17-C19), 1/100 (group C,C20-C22) and 1/1000 (group D, C23-C25) in sterile PBS,respectively. Serum samples were taken at days 1 to7 and 21; they were tested for SBV RNA and SBV-specific antibodies as described above.ResultsComparison of cell culture supernatant and infectiousserum as inoculumIn the samples taken 2 days after infection, all cattle, ei-ther inoculated with infectious serum (group 1) orculture-grown virus (group 2), scored positive in the RT-qPCR for the first time. SBV genome remained detect-able until day 5 in two out of four animals in group 1,until day 6 in the remaining two calves in group 1 andthe three animals in group 2, whereas in the last calf ingroup 2 SBV genome was detected until day 7 (Figure 1).At peak RNAemia (4 dpi), there was no significant dif-ference between the SBV RNA loads in groups 1 and 2(p = 0.14, power = 0.519).SBV RNA was not detected in the control animals atany time. Fever was not observed in any animal, but C08developed mild diarrhea for two days.Necropsy of the animals did not reveal any significantgross pathomorphological lesions. The mesenteric lymphnodes of C03 and C06-C08 were positive in the RT-qPCR (average Cq value: 34.8 ± 5.2). In addition, SBVRNA was found in the mandibular lymph nodes of C04and C06 (Cq values: 39.0, 37.8), and the spleens of C05and C06 (Cq values: 44.5, 36.9).All cattle in group 1 and 2 were positive in the ELISAtwo weeks after infection, while all in-contact animalsremained negative until the end of the study (data notshown).In vivo titration of the infectious serumIn the in vivo titration experiment, both animals inocu-lated with the undiluted serum (C15/C16), as well asC17 (group B, 1/10), scored positive in the RT-qPCR be-tween day 2 and 5. In C18 and C19 SBV genome was de-tectable for the first time on day 3. Animals in group C(1/100) scored positive on days 2 to 6, 4 to 7 and3 to 7, respectively. In the group inoculated with dilution1/1000 only one (C25) out of three animals was positive(Figure 2). Hence, the undiluted serum contained at leastFigure 2 Real-time RT-PCR results of cattle in the in-vivotitration experiment. All animals inoculated with the undilutedserum, dilution 1/10 and 1/100 were positive for several days. Onlyone (C25) out of three animals inoculated with dilution 1/1000scored positive.Wernike et al. Veterinary Research 2012, 43:84 Page 3 of 4http://www.veterinaryresearch.org/content/43/1/84102.83 cattle infectious doses per mL (calculated bySpearman-Karber method). All cattle with positive RT-qPCR results (groups A, B, C and C25) seroconverted inthe ELISA (data not shown). The mesenteric lymphnodes of these animals were positive in the RT-qPCR aswell (average Cq value: 32.1 ± 2.4). Additionally, SBVRNA was detected in the mandibular lymph nodes ofC16 C20, C22 and C25 (36.3 ± 3.3), the spleens of C17,C20 and C22 (39.6, 37.7, 44.8) and the tonsil of C19(38.1).DiscussionThe reliable induction of viremia is an important featureof challenge models for vector-borne diseases [9]. Whenviruses are passaged in cell culture, however, the positiveselection of spontaneous mutations that promote repli-cation in this environment can lead to changes in virusphenotype, including a reduced replication efficiencyand virulence in the natural hosts. This has beendemonstrated for several bunyaviruses [10,11] and iscommonly exploited for the production of AKAV vac-cines [12]. The genome stability of vector-borne virusesgrown in culture can be promoted by switching betweencell lines of mammalian and insect origin (mimickingnatural host alternation) [13], but pertinent data forbunyaviruses are not available. In the present study, thereplication of culture-grown SBV in cattle was reducedcompared to a strain that had only been passaged in thenatural host. While not statistically significant within theconstraints of this experiment (small group size, highvariability within groups), this difference provides a clearindication of the pitfalls of culture-based production ofchallenge inocula. The high level of infectivity in thetested serum batch offsets one of the major advantagesof culture-grown viruses, namely the easier availabilityof large amounts of material. Based on our findings,cattle-derived infectious serum is a viable and robust op-tion for a standardized SBV infection model, which isrequired for vaccine evaluation and pathogenesis studies.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsConceived and designed the experiments: KW ME BH MB. Performed theexperiments: KW ME AB BH. Analyzed the data: KW ME AB. All authors readand approved the final manuscript.AcknowledgementsPatrick Zitzow and Anja Landmesser rendered outstanding technicalassistance. Dedicated animal care was provided by the staff of the isolationunit of the FLI. We are also grateful to Matthias Lenk from the cell culturecollection for veterinary medicine of the FLI for his continuous support.This work was financially supported by Boehringer Ingelheim Pharma GmbH& Co. KG.Author details1Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10,Greifswald, Insel Riems 17493, Germany. 2Department of ExperimentalAnimal Facilities and Biorisk Management, Friedrich-Loeffler-Institut (FLI),Suedufer 10, Greifswald, Insel Riems 17493, Germany. 3Faculty of VeterinaryMedicine, University of Calgary, Calgary, Canada. 4Present address:Department of Veterinary Pathology, Faculty of Veterinary Medicine, FreieUniversität Berlin, Berlin, Germany.Received: 18 September 2012 Accepted: 14 November 2012Published: 11 December 2012References1. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier H,Eschbaumer M, Goller KV, Wernike K, Fischer M, Breithaupt A,Mettenleiter TC, Beer M: Novel orthobunyavirus in cattle, europe, 2011.Emerg Infect Dis 2012, 18:469–472.2. Goller KV, Höper D, Schirrmeier H, Mettenleiter T, Beer M: Schmallenbergvirus as possible ancestor of shamonda virus. Emerg Infect Dis 2012,18:1644–1646.3. 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Griot C, Gonzalez-Scarano F, Nathanson N: Molecular determinants of thevirulence and infectivity of California serogroup bunyaviruses. Annu RevMicrobiol 1993, 47:117–138.11. Lundkvist A, Cheng Y, Sjolander KB, Niklasson B, Vaheri A, Plyusnin A: Cellculture adaptation of Puumala hantavirus changes the infectivity for itsnatural reservoir, Clethrionomys glareolus, and leads to accumulationof mutants with altered genomic RNA S segment. J Virol 1997,71:9515–9523.12. Ogawa Y, Kato K, Tohya Y, Akashi H: Characterization of temperature-sensitive Akabane virus mutants and their roles in attenuation. Arch Virol2007, 152:1679–1686.13. Moutailler S, Roche B, Thiberge JM, Caro V, Rougeon F, Failloux AB: Hostalternation is necessary to maintain the genome stability of rift valleyfever virus. PLoS Negl Trop Dis 2011, 5:e1156.doi:10.1186/1297-9716-43-84Cite this article as: Wernike et al.: Schmallenberg virus challenge modelsin cattle: infectious serum or culture-grown virus?. 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