Schmallenberg virus in Culicoides spp. biting midges, the Netherlands, 2011.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

Faeces as a novel material to estimate lyssavirus prevalence in bat populations.

Rabies is caused by infection with a lyssavirus. Bat rabies is of concern for both public health and bat conservation. The current method for lyssavirus prevalence studies in bat populations is by oral swabbing, which is invasive for the bats, dangerous for handlers, time-consuming and expensive. In many situations, such sampling is not feasible, and hence, our understanding of epidemiology of bat rabies is limited. Faeces are usually easy to collect from bat colonies without disturbing the bats and thus could be a practical and feasible material for lyssavirus prevalence studies. To further explore this idea, we performed virological analysis on faecal pellets and oral swabs of seven serotine bats (Eptesicus serotinus) that were positive for European bat 1 lyssavirus in the brain. We also performed immunohistochemical and virological analyses on digestive tract samples of these bats to determine potential sources of lyssavirus in the faeces. We found that lyssavirus detection by RT-qPCR was nearly as sensitive in faecal pellets (6/7 bats positive, 86%) as in oral swabs (7/7 bats positive, 100%). The likely source of lyssavirus in the faeces was virus excreted into the oral cavity from the salivary glands (5/6 bats positive by immunohistochemistry and RT-qPCR) or tongue (3/4 bats positive by immunohistochemistry) and swallowed with saliva. Virus could not be isolated from any of the seven faecal pellets, suggesting the lyssavirus detected in faeces is not infectious. Lyssavirus detection in the majority of faecal pellets of infected bats shows that this novel material should be further explored for lyssavirus prevalence studies in bats.

PCR diagnostics: In silico validation by an automated tool using freely available software programs.

PCR diagnostics are often the first line of laboratory diagnostics and are regularly designed to either differentiate between or detect all pathogen variants of a family, genus or species. The ideal PCR test detects all variants of the target pathogen, including newly discovered and emerging variants, while closely related pathogens and their variants should not be detected. This is challenging as pathogens show a high degree of genetic variation due to genetic drift, adaptation and evolution. Therefore, frequent re-evaluation of PCR diagnostics is needed to monitor its usefulness. Validation of PCR diagnostics recognizes three stages, in silico, in vitro and in vivo validation. In vitro and in vivo testing are usually costly, labour intensive and imply a risk of handling dangerous pathogens. In silico validation reduces this burden. In silico validation checks primers and probes by comparing their sequences with available nucleotide sequences. In recent years the amount of available sequences has dramatically increased by high throughput and deep sequencing projects. This makes in silico validation more informative, but also more computing intensive. To facilitate validation of PCR tests, a software tool named PCRv was developed. PCRv consists of a user friendly graphical user interface and coordinates the use of the software programs ClustalW and SSEARCH in order to perform in silico validation of PCR tests of different formats. Use of internal control sequences makes the analysis compliant to laboratory quality control systems. Finally, PCRv generates a validation report that includes an overview as well as a list of detailed results. In-house developed, published and OIE-recommended PCR tests were easily (re-) evaluated by use of PCRv. To demonstrate the power of PCRv, in silico validation of several PCR tests are shown and discussed.

Improved PCR diagnostics using up-to-date in silico validation: An F-gene RT-qPCR assay for the detection of all four lineages of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a globally significant disease of small ruminants caused by the peste des petits ruminants virus (PPRV) that is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO). Critical to the eradication of PPR are accurate diagnostic assays. RT-qPCR assays targeting the nucleocapsid gene of PPRV have been successfully used for the diagnosis of PPR. We describe the development of an RT-qPCR assay targeting an alternative region (the fusion (F) gene) based on the most up-to-date PPRV sequence data. In silico analysis of the F-gene RT-qPCR assay performed using PCRv software indicated 98% sensitivity and 100% specificity against all PPRV sequences published in Genbank. The assay indicated the greatest in silico sensitivity in comparison to other previously published and recommended PPRV RT-qPCR assays. We evaluated the assay using strains representative of all 4 lineages in addition to samples obtained from naturally and experimentally-infected animals. The F-gene RT-qPCR assay showed 100% diagnostic specificity and demonstrated a limit of detection of 10 PPRV genome copies per µl. This RT-qPCR assay can be used in isolation or in conjunction with other assays for confirmation of PPR and should support the global efforts for eradication.