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. 2021 Feb 16;11:3866. doi: 10.1038/s41598-021-83323-9

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

Kamonpan Charoenkul 1,2, Chanakarn Nasamran 1,2, Taveesak Janetanakit 1,2, Supassama Chaiyawong 1,2, Napawan Bunpapong 1,3, Supanat Boonyapisitsopa 1, Ratanaporn Tangwangvivat 1, Alongkorn Amonsin 1,2,✉
PMCID: PMC7887266  PMID: 33594165

Abstract

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.

Subject terms: Virology, Pathogens

Introduction

Parainfluenza virus (PIV) is an enveloped, nonsegmented, single-stranded RNA virus. PIV-5 belongs to the family Paramyxoviridiae, genus Rubulavirus. The virus consists of seven genes encoding 8 proteins (F, HN, SH, M, NP, V, P, and L)1. PIV can be classified into 5 types, designated PIV 1–5. PIV-1 to PIV-4 can cause upper and lower respiratory tract infections in humans, especially in infants and young children2–5. PIV-5 has been reported to infect and cause respiratory disease in several host species.

PIV-5 was first isolated in 1956 from rhesus and cynomologus monkey kidney-cells6. The virus was previously named simian virus type 5 (SV-5) according to the host of isolation. Then, SV-5 was renamed to PIV-5 and prefixed according to the isolated species7. To date, the disease caused by PIV-5 in humans are still unclear. Some studies revealed that a virus serologically related to PIV-5 was associated with multiple sclerosis (MS), sclerosing panencephalitis (SSPE), Creutzfeldt-Jakob disease (CJD), pemphigus, atherosclerosis, Paget’s disease, hepatitis and common cold in humans8–10. There were in vitro studies and need to be identified as such PIV-5 was found in human respiratory cells and might impact human respiratory diseases11,12.

PIV-5 has been reported in several host species including pigs, cattle, dogs, hamsters, ferrets, monkeys, calves, lesser pandas and guinea pigs10,13,14. In pigs, PIV-5 co-infects with porcine reproductive and respiratory syndrome (PRRSV) and causes respiratory symptoms. In cattle, PIV-5 possibly causes severe respiratory illness and leads to a high morbidity rate in calves15. In dogs, canine parainfluenza virus type 5 (CPIV-5) was first isolated from dogs with respiratory signs in 1967 and was first named canine parainfluenza virus type 2 (CPIV-2) due to it causing a respiratory disease similar to that of human parainfluenza type 2 (HPIV-2)16. A subsequent study based on antigenic and sequence analyses revealed that CPIV-5 and HPIV-2 are different17. It has been reported that CPIV-5 is one of the common pathogens of canine infectious respiratory disease (CIRD). CPIV-5 causes mild to moderate respiratory illness in dogs. Dogs can develop severe clinical signs if co-infected with other respiratory viruses or bacteria18–20. In some cases, CPIV-5 can cause neurological disorders especially in puppies including encephalitis, seizures, myoclonus and posterior paresis21,22. The cross-species transmission of CPIV-5 has been reported in coyotes, ferrets and rodents23,24.

Interspecies transmission of PIV-5 between canines and humans has not been reported. However, a study suggested that PIV-5 might be a potential zoonotic pathogen25. Some studies have supported the hypothesis that genetic characteristics between PIV-5 isolated from canines and humans are highly similar with fewer nucleotide sequence variations (only 0.1% to 3% nucleotide difference)7,26,27. In addition, CPIV-5 can be grown in various cell lines especially human cell lines (2fTGH and HEp2) which might correlate with the host preference of the virus28. Since epidemiological and whole genome sequence information on CPIV-5 is still limited, in this study, we conducted a cross-sectional survey of CPIV-5 in dogs and characterized the whole genome of Thai CPIV-5.

Results

In this study, we investigated canine parainfluenza type 5 (CPIV-5) infection in dogs with respiratory symptoms from November 2015 to December 2018. Our results showed that 5.6% (32/571) of nasal swab samples were positive for CPIV-5. From 3 years of surveillance, the highest occurrence of CPIV-5 was observed in November 2016 (41.7%), followed by December 2016 (33.3%) with statistical significance p < 0.05 when compared to other years (Supplement Table S1). Regarding the relationship between CPIV-5 infection and age group, the occurrences of CPIV-5 was statistically more frequent in dogs < 1 year (10.0%; 24/240) than in dogs older than 5 years (3.3%; 4/120) and dogs 1–4 years (1.9%; 4/211) (p = 0.0349 and 0.0003, p < 0.05), respectively. Regarding the relationship between CPIV-5 infection and vaccination history, the occurrence of CPIV-5 infection in dogs with incomplete CPIV-5 vaccination (10.4%; 28/269) was higher than in dogs fully vaccinated (1.3%; 4/302), with statistical significance (p < 0.05).

Genetic characteristics of Thai canine parainfluenza type 5

In this study, we selected and characterized representatives of Thai CPIV-5 for whole genome sequencing (n = 3; CU-D133, CU-D151 and CU-D20804) and F, HN, V/P and SH gene sequencing (n = 10) (Table 1). Our results showed that the genome size of Thai CPIV-5 is 15,207 bp, containing seven genes as 3′-N-V/P-M-F-SH-HN-L-5′. Whole genome sequence analysis showed that Thai CPIV-5 possessed high nucleotide identity to the reference PIV5 with 96.1–99.4% nucleotide identities but low percentages of nucleotide identities with PIV-1 to PIV-4 (44.5–63.1% nucleotide identities). Comparing PIV-5, the whole genome of Thai CPIV-5 was closely related to Chinese CPIV-5 (HeN0718, 99.2% nucleotide identities) and Korean CPIV-5 (D277 and 08-1990, 99.4% and 99.2% nucleotide identities) (Table 2). For phylogenetic analysis, Thai CPIV-5 (n = 3) was grouped with PIV-5 from humans, pigs, dogs, lesser panda, and pangolins but separated from clusters of PIV-1 to PIV-4. The phylogenetic tree of the whole genome of PIV-5 could be divided into subgroups, e.g., human and simian subgroup, cattle and swine subgroup and canine subgroup. Thai CPIV-5 was grouped in the canine subgroup with CPIV-5 from China (HeN0718) and Korea (D277 and 08-1990) (Fig. 1).

Table 1.

Description of canine parainfluenza type 5 (CPIV-5) characterized in this study.

Virus Collection date Age Breed Vaccination history CPIV-5 detection Sequencing # GenBank
CU-D58 Jan 16 3 mts Siberian Husky I + F, HN, SH, V/Pa MT604002-05
CU-D103 Feb 16 2 mts Bully I + F, HN, SH, V/P MT604006-09
CU-D133 Apr 16  > 7 years Golden retriever C + WGSb MT603999
CU-D151 May 16 3 mts Pomeranian I + WGS MT604000
CU-D373 Nov 16 3 mts Pomeranian I + F, HN, SH, V/P MT604011-13
CU-D376 Dec 16  > 1 year Mixed I + F, HN, SH, V/P MT604014-17
CU-D381 Dec 16 3 mts Pekingese I + F, HN, SH, V/P MT604018-21
CU-D399 Jan 17 4 mts Pomeranian I + F, HN, SH, V/P MT604022-25
CU-D400 Jan 17 7 mts Pomeranian I + F, HN, SH, V/P MT604026-29
CU-D406 Jan 17 3 mts Pomeranian I + F, HN, SH, V/P MT604030-33
CU-D466 Mar 17 2 mts Mixed I + F, HN, SH, V/P MT604034-37
CU-D585 Sep 17 3 mts Mixed I + F, HN, SH, V/P MT604038-41
CU-D20804 Feb 18 4 mts Mixed I + WGS MT604001
CU-D361 Oct 16 2 mts Pomeranian I + − −
CU-D369 Nov 16 6 mts Pomeranian I + − −
CU-D370 Nov 16 10 years Shih-Tzu C + − −
CU-D371 Nov 16 12 years Poodle C + − −
CU-D372 Nov 16 4 mts Pomeranian I + − −
CU-D377 Dec 16  > 1 year Mixed I + − −
CU-D380 Dec 16 10 mts Mixed I + − −
CU-D390 Dec 16  > 5 years Mixed C + − −
CU-D483 Mar 17  > 1 year Mixed I + − −
CU-D489 Mar 17  > 1 year Mixed I + − −
CU-D493 Mar 17 6 mts Mixed I + − −
CU-D497 Mar 17 6 mts Mixed I + − −
CU-D20273 Dec 17 2 mts Pomeranian I + − −
CU-D20277 Dec 17 2 mts Bully I + − −
CU-D20364 Dec 17 3 mts Pomeranian I + − −
CU-D20384 Jan 18 3 mts Mixed I + − −
CU-D20803 Feb 18 3 mts Mixed I + − −
CU-D21496 May 18 2 mts Mixed I + − −
CU-D22309 Sep 18 3 mts Samoyed I + − −
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C complete vaccination, I incomplete vaccination.

aF, HN, SH, V/P; F, HN, SH, V/P gene sequencing.

bWGS; whole genome sequencing.

Table 2.

Pairwise comparison of whole genome nucleotide sequences of Thai CPIV-5 (CU-D151) with reference parainfluenza viruses.

Virus Accession no. Host Location (%) Nucleotide identity
WGS N (1530 nt) F (1590–1656 nt) HN (1698 nt) SH (135 nt) V (669 nt) P (1177 nt) M (1134 nt) L (6768 nt)
CU-D151 This study Canine Thailand 100.0 100.0 100.0 100.0 (−) 100.0 100.0 100.0 100.0
CU-D133 This study Canine Thailand 99.1 99.3 99.0 99.5 (−) 99.0 98.9 99.4 99.3
CU-D20804 This study Canine Thailand 99.2 99.5 99.4 98.8 (−) 99.3 99.2 99.1 99.2
CU-D58 This study Canine Thailand (−) 99.5 99.5 99.2 (−) 99.3 99.2 (−) (−)
CU-D103 This study Canine Thailand (−) 99.3 99.0 99.6 (−) 98.8 98.8 (−) (−)
CU-D373 This study Canine Thailand (−) (−) 99.2 97.2 (−) 99.0 99.1 (−) (−)
CU-D376 This study Canine Thailand (−) (−) 97.3 99.9 (−) 99.3 99.2 (−) (−)
CU-D381 This study Canine Thailand (−) (−) 99.5 99.8 (−) 99.3 99.2 (−) (−)
CU-D399 This study Canine Thailand (−) (−) 96.8 98.8 (−) 97.2 97.0 (−) (−)
CU-D400 This study Canine Thailand (−) (−) 99.1 99.2 (−) 99.0 99.1 (−) (−)
CU-D406 This study Canine Thailand (−) (−) 99.5 99.4 (−) 99.3 99.2 (−) (−)
CU-D466 This study Canine Thailand (−) (−) 99.3 99.5 (−) 99.1 99.2 (−) (−)
CU-D585 This study Canine Thailand (−) (−) 99.3 100.0 (−) 99.1 99.2 (−)
Reference PIV-5
AGS KX060176 AGS cell USA 96.1 96.2 95.0 95.8 (−) 95.7 95.9 95.6 96.7
DEN JQ743322 Human UK 96.6 96.6 95.6 96.4 (−) 96.1 96.1 96.0 97.0
MIL JQ743326 Human UK 96.5 96.5 95.6 96.3 (−) 96.0 96.0 96.0 97.0
MEL JQ743325 Human UK 96.5 96.3 92.5 96.4 (−) 96.1 96.1 95.9 97.0
RQ JQ743327 Human UK 96.5 96.5 95.5 96.3 (−) 96.0 96.0 95.9 97.0
LN JQ743324 Human UK 96.5 97.2 95.5 96.3 (−) 96.0 96.0 95.9 97.0
W3A JQ743318 Macaque cell USA 97.0 97.3 92.0 96.9 (−) 96.9 96.7 95.9 97.6
HeN0718 KY114804 Canine China 99.2 99.5 99.3 96.9 (−) 98.8 99.1 99.0 99.3
CC-14 KP893891 Canine China 97.2 97.5 96.4 97.6 (−) 96.7 96.9 96.3 97.8
H221 JQ743323 Canine UK 97.5 97.5 96.7 97.9 (−) 97.6 97.2 96.9 98.1
78524 JQ743319 Canine UK 97.5 97.4 96.7 97.9 (−) 97.3 97.1 96.9 97.9
CPI +  JQ743321 Canine USA 96.7 96.4 95.6 96.9 (−) 96.3 96.3 95.9 97.3
CPI- JQ743320 Canine USA 96.7 96.4 95.5 96.9 (−) 96.0 96.2 95.9 97.3
08-1990 KC237063 Canine Korea 99.2 99.5 99.5 99.5 (−) 99.4 99.3 99.5 99.4
D277 KC237065 Canine Korea 99.4 99.9 99.5 99.6 (−) 99.6 99.6 99.6 99.6
1168-1 KC237064 Canine Korea 97.4 97.1 96.6 97.9 (−) 97.0 96.9 96.9 98.0
SER JQ743328 Swine Germany 97.2 97.3 96.4 97.6 (−) 96.7 96.9 96.4 97.7
KNU-11 KC852177 Swine Korea 97.0 96.9 96.3 97.3 (−) 96.1 96.3 96.0 97.6
PV5-BC14 KM067467 Calve China 97.2 97.1 96.4 97.5 (−) 96.6 96.7 96.4 97.7
ZJQ-221 KX100034 Lesser panda China 97.3 96.9 96.5 97.6 (−) 97.0 96.9 96.7 97.9
Other reference PIV I to IV
HPIV-1 KF530221 Human Australia 45.9 (−) (−) (−) (−) (−) (−) (−) (−)
S033N JX857410 Swine Hong Kong 44.5 (−) (−) (−) (−) (−) (−) (−) (−)
HPIV-2 NC003443 Human Japan 63.1 (−) (−) (−) (−) (−) (−) (−) (−)
HPIV-3 NC001796 Human Australia 46.8 (−) (−) (−) (−) (−) (−) (−) (−)
Texas-81 EU439429 Swine USA 45.8 (−) (−) (−) (−) (−) (−) (−) (−)
HPIV-4 KF483663 Human Denmark 52.1 (−) (−) (−) (−) (−) (−) (−) (−)
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Figure 1.

Figure 1

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Phylogenic tree of the whole genome of Thai CPIV-5 and reference PIV1-5. Pink circles indicate Thai CPIV-5 in this study. The phylogenetic tree was constructed by using MEGA v.7.0 (Tempe, AZ, USA) with the neighbor-joining method with the Kimura 2-parameter with 1000 bootstrap replication52. The drawing was created by professional science figure service (BioRender.com).

Pairwise comparison of nucleotide sequences showed that the HN, F, V/P and SH genes of Thai CPIV-5 possessed high nucleotide identities to Chinese CPIV-5 (HeN0718; 96.9–99.5%) and Korean CPIV-5 (D277 and 08-1990; 99.3–99.9%), which were similar to the whole genome sequences (Table 2). The phylogenetic analysis of the F, HN, and V/P genes showed that Thai CPIV-5 was grouped with Chinese CPIV-5 (HeN0718) and Korea CPIV-5 (D277 and 08-1990) (Fig. 2). Moreover, the M, NP and L genes of Thai CPIV-5 (CU-D131, CU-D151 and CU-D20804) had the highest nucleotide identities to Korean CPIV-5 (D277; 99.6–99.9%). The phylogenetic analysis results showed that the M, NP and L genes were also closely related to CPIV-5 from Chinese and Korean strains (Fig. 2).

Figure 2.

Figure 2

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Phylogenetic trees of the HN, F, M, V/P, NP, and L genes of Thai CPIV-5 and reference PIV-1-5. Pink circles indicate Thai CPIV-5 in this study. The phylogenetic tree was constructed by using MEGA v.7.0 (Tempe, AZ, USA) with the neighbor-joining method with the Kimura 2-parameter with 1000 bootstrap replication52.

Genetic analysis of the HN gene (1698 nucleotides, 565 amino acids) of Thai CPIV-5 showed that amino acid residues at the receptor binding site (positions 186–190) and cleavage site (positions 390 and 523) of the HN protein contained QDHVS (186–190), E390 and Y523. Amino acid residues at the stalk regions contained S60, Y77, L90, E91 and Q102 identical to the reference PIV-5. Amino acid residues at positions 37, 342, 437, and 457, which correlated with neutralizing antibodies, contained E37, K342, T437, and F457. It is noted that Q342K was only observed in all Thai CPIV-5, which was identical to CPIV-5 from China (HeN0718) and Korea (D277 and 08-1990) but not in other CPIV-5 and human PIV-5 (Table 3). Amino acid residues related to host preference (human specific) at I22L, A49S, R57G, T254A, N318S, K460T and M536T were analyzed. Thai CPIV-5 contained I22, A49, R57, T254, N318, K460 and M536, which are not human specific amino acids. Unique amino acids for Thai, Chinese and Korean CPIV-5 were also observed at T19I, K43E, T62I, T141A, F252L, F353L and G446R suggesting unique subclustered characteristics (Table 4).

Table 3.

Genetic analysis of the HN gene of Thai CPIV-5 and reference PIV-5 at the receptor binding, cleavage site and stalk region.

Virus Host HN gene
HN gene Receptor binding site Cleavage site HN stalk
37 342 437 457 186–190 390 523 60 77 90 91 102
Reference PIV-5
AGS AGS cell E K T A QDHVS E Y S Y L E H
W3A Macaque cell E Q T F QDHVS E Y S Y L E Q
DEN Human E Q T F QDHVS E Y S Y L E Q
MIL Human E Q T F QDHVS E Y S Y L E Q
MEL Human E Q T F QDHVS E Y S Y L E Q
RQ Human E Q T F QDHVS E Y S Y L E Q
LN Human E Q T F QDHVS E Y S Y L E Q
SER Swine E Q T F QDHVS E Y S Y L E Q
KNU-11 Swine E Q T F QDHVS E Y S Y L E Q
PV5-BC14 Calve E Q T F QDHVS E Y S Y L E Q
ZJQ-221 Lesser panda E Q T F QDHVS E Y S Y L E Q
CAN Pangolin E Q T F QDHVS E Y S Y L E Q
H221 Canine E Q T F QDHVS E Y S Y L E Q
78524 Canine E Q T F QDHVS E Y S Y L E Q
CPI +  Canine E Q T F QDHVS E Y S Y L E Q
CPI- Canine E Q T F QDHVS E Y S Y L E Q
08-1990 Canine E K T F QDHVS E Y S Y L E Q
D277 Canine E K T F QDHVS E Y S Y L E Q
1168-1 Canine E Q T F QDHVS E Y S Y L E Q
CC-14 Canine E Q T F QDHVS E Y S Y L E Q
HeN0718 Canine E K T F QDHVS E Y S Y L E Q
This study
CU-D58 Canine E K I F QDHVS E Y S Y L E Q
CU-D103 Canine E K T F QDHVS E Y S Y L E Q
CU-D133 Canine E K T F QDHVS E Y S Y L E Q
CU-D151 Canine E K T F QDHVS E Y S Y L E Q
CU-D373 Canine E K T F QDHVS E Y S Y L E Q
CU-D376 Canine E K I F QDHVS E Y S Y L E Q
CU-D381 Canine E K T F QDHVS E Y S Y L E Q
CU-D399 Canine E Q T F QDHVS E Y S Y L E Q
CU-D400 Canine E K T F QDHVS E Y S Y L E Q
CU-D406 Canine E K T F QDHVS E Y S Y L E Q
CU-D466 Canine E K T F QDHVS E Y S Y L E Q
CU-D585 Canine E K T F QDHVS E Y S Y L E Q
CU-D20804 Canine E K T F QDHVS E Y S Y L E Q
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Table 4.

Genetic analysis of the HN gene of Thai CPIV-5 and reference PIV-5 at the human-specific residues.

Virus Host Location Primate specific amino acid Lineage specific amino acida
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 F F G
RQ Human UK L S G A S T T T K T T F F G
LN Human UK L S G A S T T T K T T F F G
W3A Macaque cell I A R A N T M T K T T F F G
SER Swine Germany I A R T N K M T K T T F F G
KNU-11 Swine South Korea I A R T N K M T K T T F F G
PV5-BC14 Calve China I A R T N K M T K T T F F G
ZJQ-221 Lesser panda China I A R T N K M T K T T F F G
CAN Pangolin China I A R T N K M T K T T F F G
H221 Canine UK I A R T N K I T K T T F F G
78524 Canine UK I A R T N K M T K T T F F G
CPI +  Canine USA I A R T N K M T K T T L F G
CPI- Canine USA I A R T N K M T K T T L F G
08-1990 Canine South Korea I A R T N K M I E I A L L R
D277 Canine South Korea I A R T N K M I E I A L L R
1168-1 Canine South Korea I A R T N K M T K T T F F G
CC-14 Canine China I A R T N K I T K T T F F G
HeN0718 Canine China I A R T N K M I E I A L L R
This study
CU-D58 Canine Thailand I A R T N K M I E I A L L R
CU-D103 Canine Thailand I A R T N K M I E I A L L R
CU-D133 Canine Thailand I A R T N K I I E I A L L R
CU-D151 Canine Thailand I A R T N K M I E I A L L R
CU-D373 Canine Thailand I A R T N K M I E I A L L R
CU-D376 Canine Thailand I A R T N K M I E I A L L R
CU-D381 Canine Thailand I A R T N K M I E I A L L R
CU-D399 Canine Thailand I A R T N K M T K T I F F G
CU-D400 Canine Thailand I A R T N K M I E I A L L R
CU-D406 Canine Thailand I A R T N K M I E I A L L R
CU-D466 Canine Thailand I A R T N K M I E I A L L R
CU-D585 Canine Thailand I A R T N K M I E I A L L R
CU-D20804 Canine Thailand I A R T N K M I E I A L L R
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aLineage: CPIV-5 sublineage; Thai, Chinese, and Korean sublineages.

Genetic analysis of the F gene showed a low level of genetic variation. Amino acid residues related to host preference (human specific) were observed at T3I, S19G, I301M, T438S, L498F, S530Q and R536Q. One Thai CPIV-5 (CU-D151) contained R536Q similar to some human PIV-5 (DEN, MIL, RQ, and LN). Moreover, Thai CPIV-5 contained 22P and 443P, which were similar to PIV-5 from humans and pigs suggesting potential human preference characteristics29–31 (Supplement Table S2). Genetic analysis of the V/P gene showed that amino acids related to viral RNA synthesis contained S157, T286 and K254 similar to most CPIV-5 (Supplement Table S3).

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).

Figure 3.

Figure 3

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Alignment of deduced amino acids of the SH gene of Thai CPIV-5 and reference PIV-5 viruses. The box indicates amino acid substitution at the stop codon (Q). Pink circles indicate Thai CPIV-5 in this study.

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 reported32. 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 groups33–35. 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 infection36. 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-537. Moreover, all three Thai-CPIV-5 contained unique amino acids of Asian CPIV-5 subcluster and distinguished from W3A and CPIV-5 from the US and UK. Thus, it more likely that the CPIV-5 in this study were isolated from naturally infected dogs in Thailand.

To date, only nine whole genome sequences of CPIV-5 are available in the GenBank database. This study provided additional information on the whole genome sequences of CPIV-5 from Thailand (n = 3). Based on phylogenetic analysis of the whole genome, Thai CPIV-5 belongs to parainfluenza type 5 and subcluster CPIV-5 (canine sublineage) and is separated from swine and human sublineage. Within the canine sublineage, Thai CPIV-5 was closely related to CPIV-5 from South Korea (08-1990 and D227) and China (CPIV-HeN0718). Thai CPIV-5 had the highest nucleotide identities (99.4%) to CPIV-5 from Korea. Phylogenetic analyses of the HN, F, V/P, M, NP and L genes showed similar results, in which Thai CPIV-5 was grouped together with CPIV-5 from Korea (08-1990 and D227) and China (CPIV-HeN0718). One Thai CPIV-5 (CU-D399) was closely related to PIV-5 from the pangolin (CAN) and lesser panda (ZJQ-221), which was similar to CPIV-5 (1168-1 from Korea). Our results suggested that Thai CPIV-5 potentially originated from the same ancestor as CPIV-5 from China and South Korea. Similarly, a unique cluster of CPIV-5 from dog in China (CC-1A, 2000s), PIV-5 from calf in China (PV5-BC14, 2014) and PIV-5 from pig in Germany (SER, 1998) and South Korea (KUN-11, 2011) was observed suggesting potential common ancestor of these viruses and required further investigations.

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 pathway38,39. Amino acid residues at L90, E91, Q102, QDHVS (186-190), E390 and Y523 are associated with viral receptor binding of the viruses40,41. Amino acid residues at K342, T437, and F457 are associated with neutralizing antibodies42. 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 cells41,43.

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 pigs29–31. 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 cells44–46. 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 protein7,31. 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 apoptosis31,47,48. It should be noted that 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.

Canine parainfluenza virus isolation

To isolate CPIV-5, RT-PCR-positive nasal swabs were subjected to virus isolation by using a Vero cell monolayer (ATCC, USA) at the Faculty of Veterinary Science, Chulalongkorn University. In brief, nasal swab sample were filtered with a 0.22 µm filter and inoculated onto a Vero cell monolayer containing Dulbecco’s minimal essential medium (DMEM, Gibco), 2% fetal bovine serum (FBS, Gibco), and gentamycin sulfate (50 μg/ml) at 37 °C in 5% CO2. If a cytopathic effect (CPE) was observed, the virus was harvested by centrifugation at 1000 rpm for 10 min. The cell suspension was then screened for CPIV-5 by using nested RT-PCR as previously described49,50. The isolated viruses were kept at − 80 °C for the pathogenesis studies in the future.

Canine parainfluenza virus characterization

In this study, Thai-CPIV-5 was selected for either whole genome sequencing (n = 3) or F, HN, V/P, and SH gene sequencing (n = 10). The representative CPIV-5 was selected based on epidemiological and demographic data such as the age of the dog, date of isolation, breed, and vaccination history. For whole genome sequencing, nucleotide sequences of each virus gene were amplified by PCR using oligonucleotide primers specific to each gene. The primers were synthesized per previous report and newly designed by using Primer 3 plus (Supplement Table S4)50,51. Nucleotide sequencing was conducted at the 1st Base Laboratories Sdn Bhd, Malaysia. The nucleotide sequences were validated and assembled by SeqMan software v.5 v.5.03 (DNASTAR Inc., Wisconsin, USA). In this study, nucleotide sequences of Thai CPIV-5 were submitted to the GenBank database under the accession numbers MT603999-MT604041 (Table 1).

Phylogenetic and genetic analyses were carried out by comparing nucleotide sequences of Thai CPIV-5 with those of PIV-5 available from the GenBank database. The reference nucleotide sequences of PIV-5 were retrieved based on geographic location, and host species including human PIV-1 (KF530221), swine PIV-1 (S033N; JX857410), human PIV-2 (NC003443), human PIV-3 (NC001796), swine PIV-3 (Texas-81; EU439429), and human PIV-4 (KF483663). Reference PIV-5 includes human strains (AGS; KX060176, DEN; JQ743322, MIL; JQ743326, MEL; JQ743325, RQ; JQ743327, LN; JQ743324), a rhesus macaque kidney cell strain (W3A; JQ743318.1), canine strains (HeN0718; KY114804, CC-14; KP893891, H221; JQ743323, 78524; JQ743319, CPI + ; JQ743321, CPI-; JQ743320, 08-1990; KC237063, D277; KC237065, 1168-1; KC237064), swine strains (SER; JQ743328, KNU-11; KC852177), a cattle strain (PV5-BC14; KM067467), a lesser panda strain (ZJQ-221; KX100034) and a pangolin strain (CAN; MH362816). Phylogenetic analysis of CPIV-5 was performed by using MEGA v.7.0 (Tempe, AZ, USA) with the neighbor-joining method with the Kimura 2-parameter with 1,000 bootstrap replicates52. For genetic analysis, the nucleotide sequences and deduced amino acids of CPIV-5 were aligned and compared using MegAlign software v.5.03 (DNASTAR Inc., Wisconsin, USA).

Statistical analysis

Categorical data corresponding to the time of sample collection, age of dogs, and vaccination history were analyzed using the Fisher's exact test (https://www.socscistatistics.com/tests/fisher). A p-value of < 0.05 was considered as statistically significant.

Ethics statement

This study was conducted under the approval of the Institute for Animal Care and Use Protocol of the CU-VET, Chulalongkorn University (IACUC # 1731074).

Supplementary Information

Supplementary Information (62.1KB, docx)

Acknowledgements

We would like to thank the Royal Golden Jubilee (RGJ) Ph.D. program, the Thailand Research Fund for supporting the first author scholarship (RGJ-PHD/0056/2557). We are grateful for the financial support from Chulalongkorn University for the research fund under the TSRI fund (CU_FRB640001_01_31_1), the 90th Anniversary Chulalongkorn University (Ratchadaphiseksomphot Endowment Fund) (GCUGR1125614077D), the Center of Excellence for Emerging and Re-emerging Infectious Diseases in Animals (CUEIDAs), and the One Health Research Cluster. The Thailand Research Fund (TRF) supported the corresponding author as a TRF Senior Scholar (RTA6080012).

Author contributions

K.C., C.N., R.T., N.B. and S.B. performed sample collection, molecular detection, whole genome characterization and analysis. C.N., S.C. and T.J. participated in whole genome sequencing and phylogenetic analysis. K.C. drafted the manuscript. A.A. (PI) designed the study, performed data analysis, drafted, revised and approved the manuscript. All authors reviewed the manuscript.

Completing interests

The authors declare no competing interests.

Footnotes

Publisher's note

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Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-021-83323-9.

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