key: cord-0764452-gqqocowm
authors: Vlasova, Anastasia N; Diaz, Annika; Damtie, Debasu; Xiu, Leshan; Toh, Teck-Hock; Lee, Jeffrey Soon-Yit; Saif, Linda J; Gray, Gregory C
title: Novel Canine Coronavirus Isolated from a Hospitalized Pneumonia Patient, East Malaysia
date: 2021-05-20
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab456
sha: 07c31a9c5846d3f13ff575c6e8c724a5a689f704
doc_id: 764452
cord_uid: gqqocowm

BACKGROUND: During the validation of a highly sensitive pan-species coronavirus (CoV) semi-nested RT-PCR assay, we found canine CoV (CCoV) RNA in nasopharyngeal swabs from eight (2.5%) of 301 patients hospitalized with pneumonia during 2017-18 in Sarawak, Malaysia. Most patients were children living in rural areas with frequent exposure to domesticated animals and wildlife. METHODS: Specimens were further studied with universal and species-specific CoV and CCoV one-step RT-PCR assays, and viral isolation was performed in A72 canine cells. Complete genome sequencing was conducted using Sanger method. RESULTS: Two of eight specimens contained sufficient amounts of CCoVs as confirmed by less-sensitive single-step RT-PCR assays, and one specimen demonstrated cytopathic effects (CPE) in A72 cells. Complete genome sequencing of the virus causing CPE identified it as a novel canine-feline recombinant alphacoronavirus (genotype II) that we named CCoV-HuPn-2018. Most of CCoV-HuPn-2018 genome is more closely related to a CCoV TN-449, while its S gene shared significantly higher sequence identity with CCoV-UCD-1 (S1 domain) and a feline CoV WSU 79-1683 (S2 domain). CCoV-HuPn-2018 is unique for a 36 nt (12-aa) deletion in the N protein and the presence of full-length and truncated 7b non-structural protein which may have clinical relevance. CONCLUSIONS: This is the first report of a novel canine-feline recombinant alphacoronavirus isolated from a human pneumonia patient. If confirmed as a pathogen, it may represent the eighth unique coronavirus known to cause disease in humans. Our findings underscore the public health threat of animal CoVs and a need to conduct better surveillance for them.

A c c e p t e d M a n u s c r i p t 5 evidence that Alphacoronavirus 1 species may infect and be associated with a clinical disease in humans. Here we report isolation, complete genome sequencing and molecular analysis of a CCoV virus from one of the pneumonia patients.

Eight of 301 nasopharyngeal swab (NPS) specimens from hospitalized pneumonia patients (2017-18 at Sibu and Kapit Hospitals, Sarawak, Malaysia) were previously confirmed to contain CCoV using a semi-nested RT-PCR assay and Sanger sequencing (Tables 1 and S1) [17] . The eight pneumonia patients all came from Sibu Hospital ( Table 1) . Seven (87.5%) were less than five years in age, four were infants, and most were from Sarawak's indigenous ethnic groups who typically live in rural/suburban longhouses or villages. Seven (87.5%) of the patients had evidence of a viral coinfection (Table 1) . All bacterial blood cultures were negative, and all patients hospitalized for 4-6 days and recovered.

RNA was extracted from suspended NPS samples using the 5X MagMAX Viral Isolation Kit (Applied Biosystems). Because one-step RT-PCR is less sensitive than nested/semi-nested RT-PCR, further characterization was conducted using one-step RT-PCR assays to ensure no contamination.

Qiagen One-step RT-PCR kit was used with the indicated primers and cycling protocols (Table S2) .

Amplicons generated with CCoV-N-F/CCoV-N-R primers were gel-extracted using QIAquick® Gel Extraction Kit (Qiagen) and sequenced using Sanger method at the Molecular and Cellular Imaging 

Canine fibroblast tumor (A72) cells (received from Dr. Alfonso Torres, Cornell College of Veterinary Medicine) were maintained and used for sample inoculation as described previously [19] . Serially diluted NPS fluids (1:10-1:10,000) were used to inoculate the A72 monolayers. After 72 hours the infected cells and medium were harvested and used for RNA extraction with the RNEasy Mini Kit (Qiagen). The immune transmission electron microscopy (I-TEM) was conducted as described previously using polyclonal anti-canine coronavirus guinea pig serum (BEI Resources, NR-2727), the I-TEM images were captured at the MCIC [20] .

The viral RNA was converted into cDNA using a SuperScript III cDNA synthesis kit (Invitrogen, USA).

Forty-two primer pairs (Table S3 ) covering the whole genome were designed based on the sequence of CCoV, strain TN-449, the most closely related strain as determined by BLASTn analysis of the partial N gene sequence of the newly identified CCoV for which the complete genome was available. Using these primers and Taq Platinum (Invitrogen) 12 amplicons (1.7-3.6 kb) were generated and purified using the QIAquick® Gel Extraction Kit and sequenced with 3× coverage using the Sanger dideoxy method with a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, USA) at the MCIC and at the James Comprehensive Cancer Center Shared Genomics Core, The Ohio State University (Columbus, OH). After the initial analysis/sequence assembly, seven additional primer pairs were designed based on the newly generated sequences to close the remaining gaps (Table S3 ). The fragments were amplified and sequenced as described A c c e p t e d M a n u s c r i p t 7 above. The 5'-and 3'-genomic ends were amplified using the 5' and 3´ RACE System for Rapid Amplification of cDNA Ends (Invitrogen) according to the manufacturer's manual.

Raw sequences were trimmed to remove low-quality reads and amplicon-primer linkers. Each ORF was analyzed using Viral Genome ORF Reader (VIGOR) to predict viral protein sequences. The annotated CCoV genome was submitted to the GenBank (accession number is MW591993). The alignments were further analyzed Sequence Manipulation Suite (SMS, Version 2) (https://www.bioinformatics.org/sms2/) to determine nt identities between the reference and newly generated sequences. Sequence alignment and phylogenetic analysis were performed using the ClustalW method and the maximum-likelihood method with the general time reversible nucleotide substitution model and bootstrap tests of 1,000 replicates of MEGAX software. The CoV genomes for reference strains from GenBank used in the phylogenetic analyses are listed in (Table 3 ). The RIP (Recombinant Identification Program; http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html) was used to identify recombination points within the CCoV-HuPn-2018 genome with the following parameters: window size of 400 and confidence threshold of 90%. Glycosylation prediction was conducted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/).

Samples from two of the eight patients from whom CCoV was earlier detected were positive in universal and CCoV-specific one-step RT-PCR assays (Table S2 ). This could be due to different quantity/integrity of CCoV in samples collected at variable time-points post infection. According to the BLASTn search, the sequences obtained for both samples using CCoV-N-F/CCoV-N-R primers M a n u s c r i p t 8 shared the highest nucleotide (nt) identity (96.31%) with several CCoV strains including TN-449 and HLJ-073 (listed in Table 3 ). We selected TN-449 sequence to design sequencing primers covering the complete genome (Table S3 ).

While eight CCoV-positive NPS samples were inoculated into A72 cells, only one sample (1153, Table 1 ) produced CPE in the cells ( Figure S1 ). The A72 cell-passaged material (P1) was inoculated into A72 cells again and CPE was observed within the same timeframe (P2). RNA extracted from both P1 and P2 tested CCoV positive; RNA extracted from P1 was used for complete genome sequencing. This virus was visualized using I-TEM ( Figure 1 ) and is referred to as CCoV-HuPn-2018 throughout (HuPn -human pneumonia).

The assembled viral genome was 29,083/29,351 (due to differences between the two 7b forms) nt Further, while the full-length S gene of CCoV-HuPn-2018 shared the highest nt identity with CCoV TN-449 (93.42%), its S1 domain was near identical to that of CCoV UCD-1 (for which only S1 sequence is available) sharing 99.19% nt identity, which was higher than for any other genomic region ( Table 3 ). The S2 domain of CCoV-HuPn-2018 shared the highest identity of 97.13% with FCoV WSU 79-1683, providing additional evidence of recombinant (feline-canine, canine-TGEV) nature of most CCoV S genes. [21] The remaining three genes encoding for structural proteins E, M and N shared the highest nt identity of 95.18%, 97.08% and 93.77%, respectively, with those of CCoV A76 (Table 3) The S protein was comprised of 1,448 aa similar to other CCoV II strains and shorter than S proteins 3 of CCoV I characterized previously [22] . Twenty-nine potential glycosylation sites were predicted in the 4 S protein of the newly identified CCoV-HuPn-2018 ( Figure S3A ) similar to other CCoVs. [22] Unlike 5

CCoV I, some FCoV and all beta-and gammacoronaviruses, the characteristic multibasic motif (RRXRR) 6 furin recognition site was absent in the S protein of CCoV-HuPn-2018, suggesting that the virus carries 7 an uncleaved S protein, similar to most other alphacoronaviruses [15] . Thus, this novel strain shares more 8 similarities with CCoV-II strains. 9

Surprisingly, there were no unique deletions or insertions in the S protein of CCoV-HuPn-2018. Also, 10 there were a total of 5 aa differences between the CCoV-HuPn-2018 and CCoV UCD-1 in the S1 domain; 11 however, these aa were identical to those found in the TGEV Purdue S1 and not unique. 12

The E protein was 81-aa long and did not contain any N-glycosylation sites, whereas three N-13 glycosylated residues have been predicted in each the 261-aa long M and the 370-aa long N proteins 14 ( Figures S3B and S3C) , which is similar to several other FCoV/CCoV strains. While no evidence of 15 recombination was observed for E, M or N proteins, the N protein contained a unique 12-aa deletion 16 within the SR-rich region (located between aa 164 and 177 for other CCoV strains). Presence of this 17 deletion was confirmed in the original NSP samples 1116 and 1153. 18

The three ORFs 3a, 3b and 3c between S and E genes encoded for proteins with sizes of 71, 71 and 19 244 aa, respectively. ORF3, previously found in CCoV I genomes only [14, 22] , was not present in the 20 new strain. The 3'-end accessory protein gene 7a encoded for 101-aa, while there were at least 2 forms of 21 7b: full-length (213-aa) and the one with a 227-nt deletion (leading to a frame shift and premature 22 truncation of the putative protein). 23

A c c e p t e d M a n u s c r i p t 13 Our previous study identified eight pneumonia patients with molecular evidence of CCoV in their 26 NPS specimens [17] . Partial sequencing and BLASTn analysis suggested that these were closely related 27 but distinct CCoV variants (Table S1 ). The eight pneumonia patients were mainly children living in long 28 houses or villages in rural/suburban areas where domestic animal and jungle wildlife exposure with the 29 family is common. 30

Here, we confirmed the presence of CCoV with different, less sensitive, one-step RT-PCR assays in 31 two specimens, grew a virus in A72 cells from one specimen, and conducted a complete genome 32 sequence analysis of the CCoV. Our results demonstrated that CCoV-HuPn-2018 is a novel canine-feline-33 like recombinant strain with a unique N. To our knowledge, this is the first report suggesting that a CCoV 34 without major genomic re-arrangements or adaptive modifications in the S protein might replicate in 35 association with pneumonia in a human host. 36

The conducted analyses demonstrated that the newly identified CCoV-HuPn-2018 was most closely 37 related to CCoV TN-449, while its S1 and S2 domains shared the highest nt identity with that of CCoV 38 UCD-1 and FCoV WSU 79-1683, respectively. These findings are suggestive of the recombinant nature 39 of this strain, similar to many previously characterized CCoVs [21] . Phylogenetic and recombinational 40 analyses confirmed that CCoV-HuPn-2018 was only distantly related to other alphacoronavirus species 41

including HCoVs (229E and NL63) and bat CoVs and likely originated via multiple recombination events 42 between different Alphacoronavirus 1 strains, but not other alphacoronaviruses. 43

The ability of the novel strain to replicate in A72 canine cells, the absence of ORF3, the higher 44 overall similarity with CCoV-II strains (TN-449 and HLJ-073) and the lack of the furin cleavage site 45 between S1 and S2 domains suggest that it belongs to CCoV genotype II [22] . 46

The unique feature not found in any other known CCoVs and Alphacoronavirus 1 species, namely the 47 12-aa deletion in the middle portion of the N protein was confirmed in both original NSP samples 1153 48 and 1116. While insertions/deletions in the N protein are not found among the known Alphacoronavirus 1 49 M a n u s c r i p t 14 strains, the deletion of the SR-rich domain within the middle region of SARS-CoV N protein reportedly 50 resulted in dramatic changes in its cellular localization soon after its zoonotic transmission [23] . Thus, 51 similar to SARS-CoV, CCoV-HuPn-2018 possesses some unique genetic features suggestive of recent 52 zoonotic transmission. Notably, such N protein re-arrangements are characteristic for SARS-CoV/SARS-53

CoV-2 with higher case-fatality rates [24] . 54

While SARS-CoV and FCoV NSP7b was not essential for viral replication in vitro and in vivo 55 experiments, its deletion/truncation may be associated with attenuated phenotype [25] . Disruption in the 56 expression of the NSPs following zoonotic transmission of SARS-CoV was reported previously 57

suggesting it may represent an adaptive mechanism [26] . Finally, deletions unique to FIPVs were found 58 in ORFs 3c and/or 7b that were hypothesized to be responsible to the shift from enteric (FCoV) to FIPV 59 phenotype and increased pathogenicity [27] . 60

The ability of CCoV to quickly evolve via frequent recombination events and induce disease of 61 variable severity is even more concerning given these data indicating that circulating CCoV may already 62 be transmittable to humans. 63

This study had a number of limitations. First, we have not met recognized standards of causality such 64 as Koch's postulates or Bradford Hill criteria. Second, we recognize that the detected CCoVs could only 65 be "carried" in some of the eight patients' airways not causing disease. However, identification of: 1) 66

FCoV-like CoVs in influenza-negative patients with acute respiratory symptoms in Arkansas and 2) 67 porcine deltacoronavirus in children in Haiti further emphasizes that Alphacoronavirus 1 species may be 68 infectious/pathogenic to humans [18, 28] . 69

In conclusion, we recovered and characterized a novel recombinant coronavirus, CCoV-HuPn-2018, M a n u s c r i p t 25 A c c e p t e d M a n u s c r i p t 30 Figure 3 

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We also thank Tea Meulia (Molecular and Cellular Imaging Center of the Ohio Agricultural Research and Development Center) for assistance with transmission electron microscopy. The studysource of NSP sampleshas received a scientific review, and all procedures followed were in accordance with the

The authors declare no conflict of interest. A c c e p t e d M a n u s c r i p t 24 *Truncated, likely non-functional, contains an out of frame 227-nt deletion close to its 5' end followed by premature stop codons, and full-length forms.