Human Norovirus Infection in Dogs, Thailand

In July 2018, recombinant norovirus GII.Pe-GII.4 Sydney was detected in dogs who had diarrhea in a kennel and in children living on the same premises in Thailand. Whole-genome sequencing and phylogenetic analysis of 4 noroviruses from Thailand showed that the canine norovirus was closely related to human norovirus GII.Pe-GII.4 Sydney, suggesting human-to-canine transmission.

https://www.qiagen.com) following the manufacturer's instructions. The virus RNA was stored at −80°C until use.
A PCR for norovirus identification was conducted as described (1,2). We use a set of oligonucleotide primers (Appendix Table 4). A 1-step reverse transcription PCR (RT-PCR) (Invitrogen, https://www.thermofisher.com) was conducted in a final volume of 25 µL containing 3 µL of template RNA, 12.5 µL of 2× reaction mixture, 0.6 µL of 10 μmol/L of forward (F4895) and reverse (R5591) primers, 1.2 µL of SuperScript III reverse transcriptase (Invitrogen), and distilled water. The RT-PCR procedure included a reverse transcription step at 55°C for 30 min; an initial denaturation step at 94°C for 2 min; followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 68°C for 1 min; and final extension step at 68°C for 6 min. To confirm the presence of noroviruses, 4 µL PCR product was subjected to electrophoresis on a 1.5% agarose gel, with RedSafe dye (Bulldog Bio, https://www.bulldog-bio.com), at 100 V for 45 min. The amplification product was visualized on a UV transilluminator. The expected size of the norovirus-positive amplified product was 493 bp.
We conducted a 1-step real-time RT-PCR for norovirus identification as described (8,9). This real-time RT-PCR was conducted by using the TaqMan Fast Virus 1-step real-time RT-PCR (Thermo Fisher Scientific, https://www.thermofisher.com) with specific primers and probe to GI and GII noroviruses was conducted in a final volume of 25 µL containing 5 µL of template RNA, 1× Master Mix, 0.25 µmol/L GI forward and reverse primers, 0.125 µmol/L of GI-JOE labeled probe, 0.25 µmol/L GII forward and reverse primers, 0.125 µmol/L of GII-FAM labeled probe, and distilled. This real-time RT-PCR included a reverse transcription step at 50°C for 10 min; an enzyme activation step at 95°C for 20 s; followed by 45 cycles of denaturation at 95°C for 3 s and annealing at 60°C for 30 s. A cycle threshold value <40 was considered as indicating GI and GII positive.

Characterization of Viruses
In this study, we selected 4 noroviruses from Thailand: including 2 from humans (CU21953 and CU21954) and 2 from dogs (CU21939 and CU21952) for whole-genome sequencing. Whole norovirus genomes were sequenced by using oligonucleotide primer sets previously described and new primer sets designed with Primer 3 Plus (Appendix Table 4) (10,11). A 25 µL RT-PCR mixture contained 3 µL of template RNA, 12.5 µL of 2× reaction mixture, 0.6 µL of 10 µmol/L forward and reverse primers, 1.2 µL of SuperScript III reverse transcriptase, and distilled water. The RT-PCR procedure included a reverse transcription step at 55°C for 30 min; an initial denaturation step at 94°C for 2 min; followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 48-55°C for 30 s, and extension at 68°C for 2 min; and a final extension step at 68°C for 6 min. Amplicons were gel-purified and sequenced (First Base Laboratories, http://www.firstbaselab.com). Nucleotide sequences were assembled and validated by using SeqMan software version 5.03 (DNASTAR Inc., https://www.dnastar.com).
Whole-genome sequences of noroviruses from Thailand were submitted to GenBank under accession nos. MK928496-9.