Elsevier

Virus Research

Volume 257, 15 September 2018, Pages 113-118
Virus Research

Widespread occurrence of squirrel adenovirus 1 in red and grey squirrels in Scotland detected by a novel real-time PCR assay

https://doi.org/10.1016/j.virusres.2018.09.007Get rights and content

Highlights

  • A real-time PCR to detect squirrel-associated adenovirus 1 (SqAdV-1) was established.

  • SqAdV-1 genomic DNA was detected in red and grey squirrels.

  • High viral genome loads were detected in intestine and liver samples of red squirrels.

  • The complete genome sequence of a SqAdV-1 strain from Scotland was determined.

  • SqAdV-1 genomes in red squirrels from Scotland and Germany are almost identical.

Abstract

The Eurasian Red Squirrel (Sciurus vulgaris) is distributed throughout large parts of Europe and Asia. However, its distribution in certain regions of Europe is endangered by the invasive, non-native Grey Squirrel (Sciurus carolinensis). Adenoviruses were already described in squirrels in Great Britain almost two decades ago. In 2013, a squirrel adenovirus (SqAdV-1) was additionally found in a red squirrel from Germany, which suffered from acute diffuse catarrhal enteritis, and the complete genome sequence was determined. Here, samples from dead red (n = 25) and grey (n = 12) squirrels collected in Scotland, UK, were analysed for the presence of this squirrel-associated virus. By using a newly developed real-time PCR targeting the adenoviral polymerase gene, viral DNA was detected in at least one of four tissue samples tested per animal in 64.0% of the red squirrels and 41.7% of the grey squirrels. Exceptionally high viral genome loads were detected in the intestine and liver, but SqAdV-1 DNA was also present in lung and kidney samples of affected animals. Almost complete genome sequence determination of a red squirrel-derived SqAdV-1 strain from Scotland indicated a very high degree of identity to the first German strain. Sequence analysis of the hexon gene, which encodes one of the major antigens of the virion, revealed an identity of 100% between viruses found in red and grey squirrels from Scotland. In conclusion, SqAdV-1 appears to be widespread in the Scottish red and grey squirrel population, which highlights the necessity for continuous wildlife surveillance. The novel real-time PCR assay offers a highly sensitive and robust method for SqAdV-1 surveillance.

Introduction

Adenoviruses are non-enveloped viruses with a virion of 70–90 nm in diameter. The genome consists of a single, linear molecule of double stranded DNA that contains inverted terminal repetitions (ITR) at both ends. A virus-coded terminal protein (TP) is covalently linked to the 5′ end of each DNA strand. The size of the genome ranges from 26 to about 48 kilo base pairs (kbp), from which about 23 to 50 different polypeptides are produced (Davison et al., 2003; Harrach et al., 2011). Commonly, adenoviruses induce no or mild clinical signs such as respiratory disease or diarrhoea, but a few produce severe systemic diseases (Hagan et al., 1988; Harrach et al., 2011).

Taxonomically, the family Adenoviridae is subdivided into the genera Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, and Siadenovirus (ICTV, 2017). Members of the genus Atadenovirus have been found in various hosts including birds, mammals and reptiles, while aviadenoviruses were identified in birds only (Ballmann and Harrach, 2016; Davison et al., 2003; Papp et al., 2009; Thomson et al., 2002; Wellehan et al., 2004). The genus Ichtadenovirus was reported from fish and members of the genus Siadenovirus have been identified in reptiles, amphibians and birds (Ballmann and Harrach, 2016; Davison et al., 2000; Kovács et al., 2003; Lee et al., 2016; Rivera et al., 2009).

Mastadenoviruses affect humans as well as a wide range of other mammals with many virus species having multiple serotypes (Hagan et al., 1988; Harrach et al., 2011). The natural host range of a specific adenovirus type is usually confined to a single species or to closely related species.

Squirrel adenoviruses (SqAdV), species Squirrel mastadenovirus A (ICTV, 2017), have been detected in a couple of European countries and Korea and the potential association with disease signs and mortality in red squirrels as a co-factor for further fatal virus infections was discussed (Abendroth et al., 2017; Duff et al., 2007; Everest et al., 2008, Everest et al., 2010a, Everest et al., 2012a, Everest et al., 2014, Everest et al., 2018; Peters et al., 2011; Romeo et al., 2014; Sainsbury et al., 2001). Historically, the vast majority of those SqAdV infections were diagnosed using transmission electron microscopy to identify virus particles in intestinal content of animals that suffered from enteritis and/or diarrhea (Duff et al., 2007; Everest et al., 2012a, 2008). Until now, only conventional PCRs specific for red squirrel adenovirus sequences have been described, one of them using a nested-PCR approach (Everest et al., 2012b; Kim et al., 2017). However, compared to such conventional PCR assays, real-time PCR systems have several advantages such as semi-quantitative analysis, the suitability for high-throughput detection and a reduced risk of cross-contamination in the absence of post-amplification handling of samples. Therefore, a Squirrel adenovirus 1 (SqAdV-1)-specific real-time PCR assay combined with an internal control system was established in the present study and was subsequently used to analyze samples from grey and red squirrels collected in Scotland.

Section snippets

Red and grey squirrel samples

Twenty-five red squirrel carcasses were collected as part of a disease surveillance scheme in which animals in Scotland are sent to the Royal (Dick) School of Veterinary Studies for post-mortem examination or were collected as part of an opportunistic health surveillance project on the Isle of Arran, in which volunteers collect red squirrels that have died from a number of causes (disease, cat-kill, road-kill, poisoning).

The 12 grey squirrels in this study came from two areas, Anwoth and

Validation of the real-time PCR

The specificity of the duplex PCR was assessed using DNA extracted from a series of lysates from cells infected with different mastadenoviruses (BAdV, PAdV, CAdV, and OAdV); all non-squirrel adenoviruses tested negative in the SqAdV-1-specific real-time PCR while the internal control assay scored positive in every case (Table 1). In addition, the SqAdV-1-specific primer and probe sequences were compared with published sequences (NCBI GenBank) with a special focus on other rodent adenoviruses,

Discussion

SqAdV infections have been described for many years and in several countries. A potential association with disease signs and mortality in red squirrels as a co-factor for further fatal virus infections has been discussed and, in this context, grey squirrels were suspected to be a potential source of infection for red squirrels in the UK (Everest et al., 2009), since they have already been identified as a reservoir for squirrelpox virus causing fatal infections in red squirrels (Rushton et al.,

Funding

This work was in part supported by the contract-research-project for the Bundeswehr Medical Service FV E/U2AD/CF512/DF557 META-InfRisk and the European ERA-NET project “EpiSeq” (grant 2811ERA094). The funders played no role in the study design, in collection, analysis, and interpretation of data and the writing of the report.

Conflicts of interest

None

Acknowledgements

The authors are very grateful to Anke Mandelkow and Caterina Fiegna for dissection of some of the squirrels, to Bianka Hillmann, Alrik-Markis Kunisch, and Patrick Zitzow for excellent technical assistance, and to Peter Garson and the Gatehouse Squirrel Group for helping to obtain samples. Non-squirrel adenoviruses were kindly provided by the virus collection of the Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

References (42)

  • K.T. Ballingall et al.

    Limited diversity associated with duplicated class II MHC-DRB genes in the red squirrel population in the United Kingdom compared to continental Europe

    Conserv. Genet.

    (2016)
  • M.Z. Ballmann et al.

    Detection and partial genetic characterisation of novel avi- and siadenoviruses in racing and fancy pigeons (Columba livia domestica)

    Acta Vet. Hung.

    (2016)
  • S. Bosch et al.

    Nachweis eines neuen Squirrel-Adenovirus bei einem Krankheitsausbruch bei Eichhörnchen (Sciurus vulgaris) in Deutschland 2013–2016

    MAUS - Mitteilungen aus unserer Säugetierwelt 20

    (2017)
  • A.J. Davison et al.

    DNA sequence of frog adenovirus

    J. Gen. Virol.

    (2000)
  • A.J. Davison et al.

    Genetic content and evolution of Adenoviruses

    J. Gen. Virol.

    (2003)
  • J.P. Duff et al.

    Enteric adenovirus infection in a red squirrel (Sciurus vulgaris)

    Vet. Rec.

    (2007)
  • D.J. Everest et al.

    Adenovirus-associated deaths in red squirrels on Anglesey

    Vet. Rec.

    (2008)
  • D.J. Everest et al.

    PCR detection of adenovirus in grey squirrels on Anglesey

    Vet. Rec.

    (2009)
  • D.J. Everest et al.

    Adenovirus in a red squirrel (Sciurus vulgaris) from Scotland

    Vet. Rec.

    (2010)
  • D.J. Everest et al.

    Retrospective detection by negative contrast electron microscopy of faecal viral particles in free-living wild red squirrels (Sciurus vulgaris) with suspected enteropathy in Great Britain

    Vet. Rec.

    (2010)
  • D.J. Everest et al.

    Adenovirus particles from a wild red squirrel (Sciurus vulgaris) from Northern Ireland

    Vet. Rec.

    (2012)
  • View full text