Lasse Dam Rasmussen

Culicoids as Vectors of Schmallenberg Virus

To the Editor: In autumn 2011, an unidentified disease of livestock was reported on both sides of the Dutch–Germany border. By using metagenomics, the etiologic agent of this disease was identified as a novel orthobunyavirus and named Schmallenberg virus (SBV) (1). Other members of the genus Orthobunyavirus (e.g., Akabane virus) are widespread in Africa and Asia; biting midges (Culicoides spp.) and mosquitoes are responsible for transmitting these viruses. Hence, we reasonably assumed that European culicoids might be responsible for transmitting SBV within Europe. We present evidence that culicoids captured October 2011 in Denmark contained SBV RNA and most likely are vectors for this agent. In autumn 2011, culicoids were collected from several sites within Denmark. One site, a chicken farm in Hokkerup (Figure A1), was selected for study because of its location close (6 km) to the German border and proximity (<10 km) to an SBV-infected sheep farm in Germany, as reported on March 9, 2012, by the Friedrich Loeffler Institute surveillance website (www.fli.bund.de). The culicoids were collected during October 14–16 by using a Mosquito Magnet Independence trap (Mosquito Magnet, Lititz, PA, USA) baited with carbon dioxide and octenol. Midges were sorted manually into 91 specimens of the C. obsoletus group (comprising C. obsoletus, C. chiopterus, C. dewulfi, and C. scoticus) and 17 of the C. punctatus sensu stricto group, then stored at −20°C. Pools of culicoids were homogenized in water (100 µL) by using a 3-mm stainless steel bead (Dejay Distribution Ltd., Launceston, UK) in a TissueLyser II (QIAGEN, Hilden, Germany) for 1 min at 25 Hz (2). After homogenization, additional water (100 µL) was added to the samples, and then the mixture was centrifuged at 3,000 × g for 5 min. Nucleic acids were extracted from the supernatant (100 µL) by using a MagNA pure LC Total Nucleic Acid Isolation Kit on a MagNA pure LC (Roche Diagnostics, Basel, Switzerland) and eluted in water (50 µL). Two separate 1-step reverse transcription quantitative PCRs (RT-qPCRs), targeting the L segment and the S segment of SBV RNA, were performed according to protocols provided by the Friedrich Loeffler Institute in Germany (1) on the extracted nucleic acids by using a Mx3005p qPCR system (Agilent Technologies, Palo Alto, CA, USA). Another RT-qPCR targeting ruminant β-actin mRNA was performed as an internal endogenous control (3). Two of 22 pools tested strongly positive for the large (L) and small (S) segments of SBV RNA. Each positive sample was derived from 5 midges of the C. obsoletus group. One pool produced cycle threshold (Ct) values of 26.4 and 24.5 (in the L segment– and S segment–specific assays, respectively), whereas the second positive pool gave Ct values of 28.8 (L segment) and 27.6 (S segment). These pools were negative for the internal endogenous control that targeted the bovine/ovine β-actin mRNA. This result makes it unlikely that the detection of SBV RNA within the midges resulted from recent blood meals from infected animals remaining within the culicoids and suggests the virus has replicated within the midges. The PCR amplicons (145 bp; Figure) from the L segment–specific RT-qPCR were sequenced by using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of 80 bp from the amplicons, excluding the primer sequences, had 100% identity with the expected region of the SBV segment L (1). Figure RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, ... Reported Ct values generated by using the same assays from blood of naturally infected cattle were 24–35 (1). Usually, ≈100 µL of bovine/ovine blood is used for virus detection, whereas <1 µL of blood remains in a midge after a blood meal. This uptake of blood should therefore lead to a Ct value that is at least 6–7 units higher (≈100-fold lower level of RNA) when a single midge is tested by RT-qPCR (4). Thus, even if all 5 culicoids in a pool had recently taken a blood meal from a viremic animal, the Ct values observed here strongly suggest replication of SBV within the C. obsoletus group midges. However, in principle, other hosts of SBV could have a much higher level of viremia than cattle and could provide the levels of SBV RNA detected. C. punctatus s.s. midges cannot be ruled out as a possible vector of SBV because of the limited number of insects tested. Our study demonstrates the presence of SBV RNA in C. obsoletus group midges caught in Denmark during October 2011. The low Ct values (i.e., high SBV RNA levels) and the absence of ruminant β-actin mRNA in these samples strongly suggest that SBV replicates in these midges and hence that the C. obsoletus group midges are natural vectors for this virus.

Development of a real-time RT-PCR assay based on primer–probe energy transfer for the detection of all serotypes of bluetongue virus

A real-time RT-PCR assay based on the primer-probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mutations in the probe region. Furthermore, melting curve analysis following immediately PCR confirms specific probe hybridization and can reveal mutations in the probe region by showing a difference in the melting point. The assay sensitivity was in the range of 10-100 target copies and the specificity tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging strains.

Bluetongue in Denmark during 2008

Following the first ever case of bluetongue in Denmark during late 2007, further outbreaks were observed in Denmark during 2008, despite vaccination against bluetongue virus (BTV) serotype 8 (BTV-8) in the southern part of the country. In total, 15 separate outbreaks of infection were identified, mostly as a result of clinical suspicions but also because of surveillance of bulk milk samples. These outbreaks led to extensions of the original vaccination zone planned for 2008. Blood samples from clinical suspects were analysed using ELISA and real-time RT-PCR assays for the presence of anti-BTV antibodies and viral RNA, respectively. A newly infected calf from the primary outbreak in 2008 was studied for a period of three months, during which time it seroconverted to BTV, but the presence of viral RNA in its blood was maintained throughout this time. Each outbreak was caused by BTV-8, as determined by a serotype-specific real-time RT-PCR assay. Furthermore, the nucleotide sequence of a portion of segment 2 of the viral RNA (encoding the outer capsid protein VP2) from the samples analysed was identical to the BTV-8 segment 2 that circulated in the Netherlands during 2006.

No evidence of enteric viral involvement in the new neonatal porcine diarrhoea syndrome in Danish pigs

BackgroundThe aim of this study was to investigate whether the syndrome New Neonatal Porcine Diarrhoea Syndrome (NNPDS) is associated with a viral aetiology. Four well-managed herds experiencing neonatal diarrhoea and suspected to be affected by NNPDS were included in a case-control set up. A total of 989 piglets were clinically examined on a daily basis. Samples from diarrhoeic and non-diarrhoeic piglets at the age of three to seven days were selected for extensive virological examination using specific real time polymerase chain reactions (qPCRs) and general virus detection methods.ResultsA total of 91.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each.ConclusionsOur detailed analyses of piglets from NNPDS-affected herds demonstrated that viruses did not pose a significant contribution to NNPDS. However, further investigations are needed to investigate if a systemic virus infection plays a role in the pathogenesis of NNPDS.