Background
Methods
Sample collection and nucleic acid preparation
Host taxonomic name (subfamily, species) | Host common name | n positive/ total n tested in this study | |
---|---|---|---|
Sciurinae | |||
Sciurus carolinensis | Eastern grey squirrel | 0/77a | 0/11a |
Sciurus variegatoides | Variegated squirrel | 7/7b | |
Sciurus vulgaris | Eurasian red squirrel | 0/77a | |
Tamiasciurus hudsonicus | American red squirrel | 0/1a | |
Xerinae | |||
Urocitellus richardsonii | Richardson’s ground squirrel | 0/11a | |
Tamias striatus | Eastern chipmunk | 0/2a | |
Callosciurinae | |||
Callosciurus erythraeus | Pallas’s squirrel | 0/35a | |
Callosciurus prevostii | Prevost’s squirrel | 10/17b | |
total | 0/126 | 17/112 |
Host taxonomic name (subfamily, species) | Host common name | Origina | Polyomavirus | Organs tested with PCRs | n tested squirrels | n tested samples | n samples positive in generic nested PCR | n samples positive in virus specific nested PCR | n animals positive in generic or specific nested PCR |
---|---|---|---|---|---|---|---|---|---|
Sciurinae | |||||||||
Sciurus carolinensis | Eastern grey squirrel | Penicuik, Scotland | ScarPyV1 | lung, spleen | 6 | 11 | 1 | 8 | 6 |
Anwoth, Scotland | lung, spleen | 3 | 6 | 0 | 0 | 0 | |||
Kirkbright, Scotland | lung, spleen | 4 | 8 | 0 | 0 | 0 | |||
Dumfries, Scotland | ScarPyV1 | spleen | 13 | 13 | n. d.b | 9 | 9 | ||
Borders Region, Scotland | ScarPyV1 | spleen | 13 | 13 | n. d. | 4 | 4 | ||
Brampton, England | ScarPyV1 | spleen | 10 | 10 | n. d. | 9 | 9 | ||
Pennsylvania, USA | ScarPyV1 | lung | 5 | 5 | 0 | 2 | 2 | ||
Piedmont Region, Italy | ScarPyV1 | lung, spleen | 30 | 45 | 0 | 1 | 1 | ||
Lomardy Region, Italy | lung, spleen | 4 | 7 | 0 | 0 | 0 | |||
subtotal | 88 | 118 | 1 | 33 | 31 (35.2%) | ||||
Sciurus vulgaris | Eurasian red squirrel | Scotland (road mortality) | lung, spleen | 7 | 12 | 0 | - c | 0 | |
Isle of Arran, Scotland | lung, spleen | 12 | 13 | 0 | – | 0 | |||
Neustadt, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Munich, Germany | lung, spleen | 16 | 22 | 0 | – | 0 | |||
Waiblingen, Germany | lung, spleen | 2 | 4 | 0 | – | 0 | |||
Heidelberg, Germany | spleen | 1 | 1 | 0 | – | 0 | |||
Bad König, Germany | lung, spleen | 2 | 4 | 0 | – | 0 | |||
Dresden, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Wentorf, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Billigheim, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Rödermark, Germany | lung, spleen | 6 | 12 | 0 | – | 0 | |||
Leutenbach, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Bonn, Germany | lung | 1 | 1 | 0 | – | 0 | |||
Bad Nauheim, Germany | lung | 1 | 1 | 0 | – | 0 | |||
Oberhausen, Germany | lung | 1 | 1 | 0 | – | 0 | |||
Berlin, Germany | lung, spleen | 4 | 8 | 0 | – | 0 | |||
Stuttgart, Germany | lung, spleen | 6 | 12 | 0 | – | 0 | |||
Görlitz, Germany | lung, spleen | 6 | 12 | 0 | – | 0 | |||
Sternenfels, Germany | lung, spleen | 7 | 13 | 0 | – | 0 | |||
subtotal | 77 | 126 | 0 | 0 | |||||
Sciurus variegatoides | Variegated squirrel | Germany | SvarPyV1 | spleen | 7 | 7 | 1 | – | 1 (14.3%) |
Tamiasciurus hudsonicus | American red squirrel | Pennsylvania, USA | lung | 1 | 1 | 0 | – | 0 | |
Xerinae | |||||||||
Urocitellus richardsonii | Richardson’s ground squirrel | Winnipeg, Canada | spleen | 11 | 11 | 0 | – | 0 | |
Tamias striatus | Eastern Chipmunk | Pennsylvania, USA | lung, spleen | 2 | 2 | 0 | – | 0 | |
Callosciurinae | |||||||||
Callosciurus erythraeus | Pallas’s squirrel | Lombardy Region, Italy | CeryPyV1 | lung, spleen | 35 | 44 | 8 | 18 | 15 (42.9%) |
Callosciurus prevostii | Prevost’s squirrel | Germany | CprePyV1 | spleen | 17 | 17 | 3 | 4 | 4 (23.5%) |
total | 238 | 326 | 13 | 55 | 51 |
Host taxonomic name (subfamily, species) | Host common name | Origina | Herpesvirus | Organs tested with PCRs | n tested squirrels | n tested samples | n samples positive in generic PCR | n samples positive in virus specific nested PCR | n animals positive in generic or specific nested PCR |
---|---|---|---|---|---|---|---|---|---|
Sciurinae | |||||||||
Sciurus carolinensis | Eastern grey squirrel | Penicuik, Scotland | ScarGHV1 | lung, spleen | 6 | 11 | 8 | n. d.b | 5 |
Anwoth, Scotland | lung, spleen | 3 | 6 | 0 | - c | 0 | |||
Kirkbright, Scotland | ScarGHV1 | lung, spleen | 4 | 8 | 2 | n. d. | 1 | ||
Dumfries, Scotland | ScarGHV1 | spleen | 13 | 13 | 2 | n. d. | 2 | ||
ScarGHV2 | spleen | 1 | n. d. | 1 | |||||
Borders Region, Scotland | ScarGHV1 | spleen | 13 | 13 | 3 | n. d. | 3 | ||
Brampton, England | ScarGHV1 | spleen | 10 | 10 | 9 | n. d. | 9 | ||
Pennsylvania, USA | ScarGHV1 | lung | 5 | 5 | 3 | n. d. | 3 | ||
ScarGHV2 | lung | 1 | 0 | 1 | |||||
Piedmont Region, Italy | ScarBHV1 | lung, spleen | 30 | 45 | 2 | 4 | 4 | ||
Lombardy Region, Italy | ScarBHV1 | lung, spleen | 4 | 7 | 0 | 1 | 1 | ||
subtotal | ScarGHV1 | 88 | 118 | 29 | n. d. | 28 (31.8%) | |||
ScarGHV2 | 88 | 118 | 2 | 0 | 2 (2.3%) | ||||
ScarBHV1 | 88 | 118 | 2 | 5 | 5 (5.7%) | ||||
Sciurus vulgaris | Eurasian red squirrel | Scotland (Scotland (road mortality)) | SvulBHV1 | lung, spleen | 7 | 12 | 9 | n. d. | 6 |
Isle of Arran, Scotland | SvulBHV1 | lung, spleen | 12 | 13 | 7 | n. d. | 7 | ||
Neustadt, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Munich, Germany | SvulBHV1 | lung, spleen | 16 | 22 | 9 | n. d. | 7 | ||
Waiblingen, Germany | lung, spleen | 2 | 4 | 0 | – | 0 | |||
Heidelberg, Germany | spleen | 1 | 1 | 0 | – | 0 | |||
Bad König, Germany | lung, spleen | 2 | 4 | 0 | – | 0 | |||
Dresden, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Wentorf, Germany | SvulBHV1 | lung, spleen | 1 | 2 | 1 | n. d. | 1 | ||
Billigheim, Germany | lung, spleen | 1 | 2 | 0 | – | 0 | |||
Rödermark, Germany | SvulBHV1 | lung, spleen | 6 | 12 | 3 | n. d. | 2 | ||
Leutenbach, Germany | SvulBHV1 | lung, spleen | 1 | 2 | 1 | n. d. | 1 | ||
Bonn, Germany | SvulBHV1 | lung | 1 | 1 | 1 | n. d. | 1 | ||
Bad Nauheim, Germany | lung | 1 | 1 | 0 | – | 0 | |||
Oberhausen, Germany | lung | 1 | 1 | 0 | – | 0 | |||
Berlin, Germany | SvulBHV1 | lung, spleen | 4 | 8 | 3 | n. d. | 2 | ||
Stuttgart, Germany | SvulBHV1 | lung, spleen | 6 | 12 | 1 | n. d. | 1 | ||
Görlitz, Germany | SvulBHV1 | lung, spleen | 6 | 12 | 2 | n. d. | 2 | ||
Sternenfels, Germany | SvulBHV1 | lung, spleen | 7 | 13 | 2 | n. d. | 1 | ||
subtotal | 77 | 126 | 39 | 31 (40.3%) | |||||
Sciurus variegatoides | Variegated squirrel | Germany | spleen | 7 | 7 | 0 | – | 0 | |
Tamiasciurus hudsonicus | American red squirrel | Pennsylvania, USA | lung | 1 | 1 | 0 | – | 0 | |
Xerinae | |||||||||
Urocitellus richardsonii | Richardson’s ground squirrel | Winnipeg, Canada | UricGHV1 | spleen | 11 | 11 | 10 | n. d. | 10 (90.9%) |
Tamias striatus | Eastern Chipmunk | Pennsylvania, USA | TstrGHV1 | lung, spleen | 2 | 2 | 2 | 2 | 2 (100.0%) |
Callosciurinae | |||||||||
Callosciurus erythraeus | Pallas’s squirrel | Lombardy Region, Italy | CeryBHV1 | lung, spleen | 35 35 | 44 | 18 | 11 | 23 (65.7%) |
CeryGHV1 | lung, spleen | 44 | 2 | 5 | 6 (14.3%) | ||||
Callosciurus prevostii | Prevost’s squirrel | Germany | CpreBHV1 | spleen | 17 17 | 17 | 1 | 1 | 1 (5.9%) |
CpreGHV1 | spleen | 17 | 1 | 1 | 1 (5.9%) | ||||
total | 238 | 326 | 104 | 27 | 108 |
Bornavirus screening of squirrel brain samples
Identification of polyomaviruses and herpesviruses with generic PCR assays
Amplification of gB sequences of betaherpesviruses and gammaherpesviruses with generic PCR
Nested long-distance PCR with virus-specific primers
Hemi-nested herpesvirus DPOL PCR
Nested PCR with polyomavirus-specific primers
Nested PCR with herpesvirus-specific primers
RT-PCR and PCR controls
PCR product purification and sequencing
Synthesis of polyomavirus early and late region
Cell lines
Transfection of cells
RNA extraction and cDNA synthesis
PCR for identification of introns
Bioinformatics and phylogenetic analysis
Results
Bornavirus analysis
Identification of novel polyomaviruses and herpesviruses
Amplification of complete polyomavirus genomes
Virus name | Country of origin | Sample ID | Sequence length (bp) | Complete genome | GenBank accession number |
---|---|---|---|---|---|
CeryPyV1 | Italy | #10239, #10271, #10275 | 5112 | + | MK671087, MK671088, MK671089 |
CprePyV1 | Germanya | #10295, #10296, #10304 | 5464 | + | MK883808, MK883809, MK883810 |
ScarPyV1 | UK | #9804, #9982, #10018 | 5236, 5237, 5237 | + | MK671101, MK671096, MK671097 |
SvarPyV1 | Germanya | #10291 | 213 | MK671090 | |
CeryBHV1 | Italy | #10257, #10259, #10262 | 478 | MK957142, MK957143, MK957144 | |
CpreBHV1 | Germanya | #10298 | 478 | MN037512 | |
ScarBHV1 | Italy | #10197 | 412 | MN047451 | |
SvulBHV1 | UK | #9807, #9808, #9813, #9824 | 3336, 3336, 3327, 3336 | MK671091, MK671092, MK671093, MK671094 | |
Germany | #9886 | 3442 | MK671095 | ||
CeryGHV1 | Italy | #10276 | 472 | MK957139 | |
CpreGHV1 | Germanya | #10305 | 166 | not archivable in GenBank (< 200 bp) | |
ScarGHV1 | UK | #9783, #9800, #9802 | 3334 | MK671098, MK671099, MK671100 | |
ScarGHV2 | USA | #10179 | 166 | not archivable in GenBank (< 200 bp) | |
UricGHV1 | Canada | #10168, #10170, #10171, #10173, #10174, #10175 | 3212 | MK671102, MK671103, MK671104, MK671105, MK671106, MK671107 | |
TstrGHV1 | USA | #10182, #10183 | 443 | MK957140, MK957141 |
Identification of splice sites in early and late regions of the novel polyomaviruses
Polyomavirus | CDSa | Splice donor site EXON|intronb | Splice acceptor site intron|EXONb |
---|---|---|---|
Sciurus carolinensis polyomavirus 1 | STAg | AAG|gttagt (94) | atggaactgcag|CA (80) |
LTAg | GAG|gtgaga (95) | cctcttaataag|AT (73) | |
VP2 | CAG|gtgagg (96) | attctcttgtag|GA (85) | |
Callosciurus prevostii polyomavirus 1 | STAg | ATG|gtgagt (93) | tacctttaacag|AT (85) |
LTAg | GAG|gtaaaa (83) | tacctttaacag|AT (85) | |
VP2 | GAG|gtaaga (96) | ttccttttgtag|GA (87) | |
Callosciurus erythraeus polyomavirus 1 | LTAg | GAG|gtacgt (89) | tgcttctttcag|GA (93) |