Molecular detection and whole genome characterization of Canine Parainfluenza type 5 in Thailand

Parainfluenza virus type 5 (PIV-5) causes respiratory infection in several animal species and humans. Canine parainfluenza virus type 5 (CPIV-5) causes respiratory disease in domestic dogs worldwide. In this study, we conducted a cross-sectional survey of CPIV-5 in dogs with respiratory symptoms from small animal hospitals in Thailand from November 2015 to December 2018. Our results showed that 32 out of 571 nasal swab samples (5.6%) were positive for CPIV-5 by RT-PCR specific to the NP gene. To characterize the viruses, three representative CPIV-5 were subjected to whole genome sequencing, and an additional ten CPIV-5 were subjected to HN, F, SH and V/P gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai CPIV-5 was closely related to the CPIV-5 isolated from China and Korea. In conclusion, this study constitutes a whole genome characterization of CPIV-5 from dogs in Thailand. The surveillance of CPIV-5 should be further investigated at a larger scale to determine the dynamics, distribution and potential zoonotic transmission of CPIV-5.

Genetic analysis of the SH gene showed that Thai CPIV-5 (CU-D58, CU-D103, CU-D133, CU-D151, CU-D376, CU-D381, CU-D406, and CU-D20804) contained a non-synonymous substitution at the start codon (M1T). Distinct nucleotide substitutions at T133C were observed and resulted in the extension of four amino acids at the stop codon, similar to those of CPIV-5 from China and Korea. Thus, the SH protein of Thai, Chinese, and Korean CPIV-5 is four amino acids longer than that of the reference PIV-5 (Supplement Table S3 and Fig. 3).

Discussion
Parainfluenza virus type 5 (PIV-5) can infect and cause respiratory diseases in various mammals. Canine parainfluenza virus type 5 (CPIV-5) is highly contagious and causes mild to moderate respiratory diseases in dogs worldwide. Coinfection with CPIV-5 and other viruses or bacteria can cause more virulent clinical signs. This study revealed the occurrence of CPIV-5, which was relatively high during the winter season in Thailand (November to January). A similar finding of high occurrence detected in the cold season has also been reported 32 . CPIV-5 could be detected in younger dogs (< 1 year) more than in older dogs. Dogs of all ages could be infected with CPIV-5, but younger dogs (< 1 year) are more susceptible. This observation is in agreement with a previous report that CPIV-5 could be observed more in younger dogs than in dogs in other age groups [33][34][35] . Regarding vaccination history, CPIV-5 infection was higher in dogs with incomplete vaccination (10.4%) than in dogs with complete vaccination (1.32%). The CPIV-5 vaccine used in Thailand was modified live CPIV-5 combined with other pathogens (e.g., canine distemper virus, canine parvovirus and canine coronavirus). Some studies have suggested that vaccinated dogs can show mild clinical signs and shed the virus after infection 36 . It is noted that, the CPIV-5 characterized in this study was obtained from nasal swabs of dogs with and without vaccination. A previous study revealed that whole genome sequences of CPIV-5 vaccine was identical with PIV-5 strain W3A, which different from Thai-CPIV-5 37 . Moreover, all three Thai-CPIV-5 contained unique amino acids of Asian   www.nature.com/scientificreports/ Thai CPIV-5 contained no amino acid mutations in the HN protein at the receptor binding site, cleavage site or HN stalk region. It has been reported that the amino acid residue at E37 is associated with virus entry into host cells by clathrin-coated pits and the endocytic pathway 38,39 . Amino acid residues at L90, E91, Q102, QDHVS (186-190), E390 and Y523 are associated with viral receptor binding of the viruses 40,41 . Amino acid residues at K342, T437, and F457 are associated with neutralizing antibodies 42 . In this study, some Thai CPIV-5 contained T437I (CU-D58 and CU-D376) and K342Q (CU-D399) which is similar to PIV-5 from dogs and humans. However, the importance of these mutations (T437I and K342Q) in neutralizing antibodies requires further investigation. A previous study reported that mutations in HN stalk regions might affect viral fusion to host cells 41,43 .
Thai CPIV-5 contained some host preference amino acid residues (human specific residues) in the F gene. For example, the amino acids at 22P and 443P in the F gene were observed in both Thai CPIV-5 and PIV-5 from humans and pigs [29][30][31] . One Thai CPIV-5 (CU-D151) also contained R536Q, similar to human PIV-5. For the V and P proteins, there was no amino acid mutation in Thai PCIV-5. It has been reported that amino acid mutations of S157F, K254 R and T286A of V and P proteins can result in high progeny virus production and the apoptosis of infected cells [44][45][46] . For the SH protein, Thai CPIV-5 contained an amino acid substitution at the start codon, which can also be observed in swine PIV-5, cattle PIV-5 and canine PIV-5. Mutation of the start codon can result in no expression of the SH protein 7,31 . The function of the SH protein is unclear, but some studies have reported an association with virus survival in host cells and control of host cell apoptosis 31,47,48 . It should be noted that Table 4. Genetic analysis of the HN gene of Thai CPIV-5 and reference PIV-5 at the human-specific residues. a Lineage: CPIV-5 sublineage; Thai, Chinese, and Korean sublineages.

Virus Host Location
Primate specific amino acid Lineage specific amino acid a 22 49 57 254 318 460 536 19 43 62 141 252 353 446   Reference PIV-5   AGS  AGS Cell  L  S  G  A  S  T  T  T  K  T  T  F  F  G   DEN  Human  UK  L  S  G  A  S  T  T  T  K  T  T  F  F  G   MIL  Human  UK  L  S  G  A  S  T  T  T  K  T  T  F  F  G   MEL  Human  UK  L  S  G  A  S  T  T  T  K  T  T  www.nature.com/scientificreports/ Thai, Chinese and Korean CPIV-5 contained four amino acids longer than the reference PIV-5. Thus, the SH gene can be used as a genetic marker for the differentiation of Asian CPIV-5 from other CPIV-5.
In summary, this study is the first report of whole genome characterization of CPIV-5 in Thailand. Phylogenetic analyses showed that Thai CPIV-5 might have originated from a common ancestor with CPIV-5 from Korea and China. To date, there is no evidence of PIV-5 cross-species transmission between dogs and humans. However, it is imperative to educate pet owners, veterinarians and others who come into close contact with domestic dogs about zoonotic awareness. In Thailand, the surveillance of CPIV-5 should be further investigated on a larger scale to determine the dynamics, distribution and genetic characteristics of CPIV-5.

Materials and methods
Canine samples. From November 2015 to December 2018, a total of 571 nasal swab samples were collected from dogs with respiratory symptoms, including sneezing, nasal discharge, cough, and dyspnea. Sample collection was conducted at Chulalongkorn University's Veterinary Teaching Hospital and private small animal hospitals in Bangkok, Thailand. The animal demographic data, including age, sex, breed, contact history, and vaccination history, were recorded. This study was conducted under approval from the Institute of Animal Use and Care Committee (IACUC# 1731074), and all procedures were completed in accordance with the relevant guidelines and regulations.
Canine parainfluenza virus identification. RNA extraction from nasal swab samples was conducted by using the QIAmp viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. Briefly, 140 μl of nasal swab sample was lysed by Buffer AVL-carrier RNA and 560 μl of ethanol. The mixture was centrifuged and transferred into a column, and then 500 μl each of buffers AW1 and AW2 were added. Finally, the RNA was eluted by 50 μl of buffer AVE. RNA was stored at − 20 °C until use. CPIV-5 detection was performed by using a nested RT-PCR assay specific to the NP gene of PIV-5 (Supplement Table S4) 49 . Briefly, one-step nested RT-PCR was conducted in a total final volume of 25 μl comprised of 3 μl of template RNA, 12.5 μl of 2 × reaction mix, 0.6 μl of 10 μM forward (CPiV-F363) and reverse primer (CPiV-R538), 1.2 μl of SuperScript III RT (Invitrogen, USA) and distilled water to a final volume of 25 μl. The first round of PCR product was diluted 1:5 with distilled water and subjected to a second round by using the TopTaq Master Mix Kit (Qiagen, Germany). The final volume was 20 μl, including 10 μl of 2 × TopTaq Master Mix, 1 μl of 10 μM forward (CPiV-F428) and reverse primer (CPiV-R538), 2 μl of 10 × coral load, and 1 μl of DNA. For the first round of nested RT-PCR conditions, the reaction contained a cDNA synthesis step at 55 °C for 30 min, an initial denaturation step at 94 °C for 2 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 68 °C for 30 s, and a final extension step at 68 °C for 6 min. For the second round of nested PCR conditions, the reaction comprised an initial denaturation step at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s and a final extension step at 72 °C for 7 min. To confirm CPIV5, 4 μl of PCR product was run on a 1.5% agarose gel with red safe. The expected size of the positive CPIV-5 product was 188 bp. Statistical analysis by the Fisher's exact test was used to compare the proportion of CPIV-5 positivity among dogs categorized by the time of sample collection, age of dogs, and vaccination history. www.nature.com/scientificreports/