key: cord-0898021-uw8oj434
authors: Bui, Christine H T; Yeung, H W; Ho, John C W; Leung, Connie Y H; Hui, Kenrie P Y; Perera, Ranawaka A P M; Webby, Richard J; Schultz-Cherry, Stacey L; Nicholls, John M; Peiris, J S Malik; Chan, Michael C W
title: Tropism of SARS-CoV-2, SARS-CoV and influenza virus in canine tissue explants
date: 2021-01-04
journal: J Infect Dis
DOI: 10.1093/infdis/jiab002
sha: f408db5a5799de83650e52aa912c7dc48b1f47eb
doc_id: 898021
cord_uid: uw8oj434

BACKGROUND: Human spillovers of SARS-CoV-2 to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns towards the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses which might result in further reassortment. METHODS: We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7 and H9 subtypes and influenza B viruses of Yamagata-like and Victoria-like lineages in ex-vivo canine nasal cavity (NC), soft palate (SP), trachea (T) and lung (L) tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues. RESULTS: There was limited productive replication of SARS-CoV-2 in canine NC and SARS-CoV in canine NC, SP and L with unexpectedly high ACE2 levels in canine NC and SP. Meanwhile, the canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors. CONCLUSIONS: Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the plenty of chances for spillover during outbreaks.

A c c e p t e d M a n u s c r i p t 4 Background Dogs are called "man's best friend", offering companionship, services, loyalty and love to their human counterparts. According to the 2019-2020 American Pet Products Association (APPA) National Pet Owners Survey, over 50% of the U.S. households own a pet dog. Many consider their dogs to be members of the family and sleep next to them on their beds [1] .

However, eating dog meat is also a norm in many countries, including China, South Korea and Vietnam.

In the context of the close and complicated relationships between dogs and humans, zoonosis and reverse zoonosis become a concern. Recently, the isolation of SARS-CoV-2 from a pet dog in Hong Kong have raised concern about the possible role of dogs in SARS-CoV-2 transmission [2] . Furthermore, the emergence of a highly contagious avian-origin H3N2 canine influenza virus in South Korea [3, 4] and China [5] and its rapid geographical expansion to places like the U.S. [6] and Canada [7] have brought fear of both the zoonotic potential of the virus and the susceptibility of dogs to a wide variety of human and avian influenza viruses which historically caused seasonal epidemics and periodic unpredictable pandemics in humans. Serological surveys of human H1N1 and H3N2 seasonal and 2009 H1N1 pandemic (2009 H1N1 pdm) viruses in pet dogs showed seroprevalence between 1.2% to 9.5% [8] [9] [10] , providing evidence to past infection. With swine being historically considered a "mixing vessel" for influenza viruses [11, 12] , any animals, including dogs, can potentially be "mixing vessels" in addition to swine if they are similarly susceptible to infection with both human and avian influenza viruses. Replication in dogs provides a chance for viruses to amplify, mutate and reassort, thus facilitating cross-species transmission and the emergence of new viruses with potential threat to public health. Therefore, understanding the ability of these viruses to replicate in dogs is important in unveiling past events and prevent future outbreaks through appropriate prevention and control methods.

A c c e p t e d M a n u s c r i p t 5 Similar to the use of ex-vivo human respiratory explant cultures to investigate viral tropism and pathogenesis [13] [14] [15] , we utilize tissue explants of canine nasal cavity (NC), soft palate (SP), trachea (T) and lung (L) to systematically risk assess the susceptibility of dogs to the infection of currently pandemic, circulating and of public health concern coronaviruses and influenza viruses, including SARS-CoV-2, SARS-CoV, human and avian influenza A viruses (IAVs) of H1, H3, H5, H7 and H9 subtypes and influenza B viruses (IBVs) of Yamagata-like and Victoria-like lineages, in an attempt to better understand the role of dogs in the epidemiology of these emerging infectious viruses.

We used: SARS-CoV-2 (BetaCoV/HongKong/VM20001061/2020 and SARS-CoV-2/canine/HKG/20-03695/2020), isolated from the nasopharyngeal aspirate and throat swab of a confirmed COVID-19 patient in Hong Kong in January, 2020, [14] 

Carcasses of apparently healthy stray/abandoned mongrel dogs of both sexes in Hong Kong were collected from the pounds shortly after being euthanized with xylazine and ketamine combination and an overdose of sodium pentobarbital injection. The dogs were not sacrificed for the purpose of our experiments but were diagnosed to have serious temperament issues or problems which could not be rehomed. The dogs were adults, but the ages were not known due to technical difficulties. Nasal and tracheal swabs were collected for the qPCR detection 

The SP was obtained from an opening at the throat. After rinsing in Washing Medium, tissues at the nasopharyngeal side was removed leaving behind the oral epithelium with minimum connective tissues. The epithelium was sectioned into square explants and put on surgical sponges as in NC explants and cultured in SP culture medium containing Ham's F-12K

(Kaighn's) Medium (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 0.1 mg/ml gentamicin (Gibco).

The middle part of the tracheal tube was excised from the respiratory tract, rinsed in Washing

Medium and cut open. The epithelium attached to cartilage was cut into square explants, put on surgical sponges and cultured in NC-T culture medium as in NC explants.

The tips of lung lobes of approximately 5 mm in depth were excised, cut into triangular sheets of around 1mm thick, rinsed with Washing Medium and cultured in L culture medium A c c e p t e d M a n u s c r i p t 8 containing Ham's F-12K (Kaighn's) Medium (Gibco), 100 U/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco).

The above methods were adopted from previous studies with modifications [13, 18, 19] .

Within 3 hours after isolation, canine explants were submerged in 1 ml of approximately 1 x assays. Experiments were performed with tissues from 3 or more dogs.

Canine tissues were fixed in 10% neutral-buffered formalin immediately after isolation and later paraffin embedded. Formalin-fixed paraffin-embedded (FFPE) tissue sections were deparaffinized in xylene and rehydrated in graded alcohols: 100%, 95%, and 80%, for subsequent processing. 

Consistent with the in-vivo challenge study of SARS-CoV-2 in beagles [20] , both human and canine isolates of SARS-CoV-2 in our study replicated poorly in the four canine explant systems ( Figure 1 ). There was limited productive replication in NC explants, with virus titres in the culture supernatant reaching 3.1 to 3.2 log TCID 50 /ml by 96 hpi, which may correspond in part with the low positive sporadic detection of SARS-CoV-2 in canine nasal swabs [2] .

Replication in other explants remained minimal or undetected. Although the two SARS-CoV-2 isolates were clearly distinguishable with seven amino acid difference (Table 1) M a n u s c r i p t 11

Staining for ACE2, the entry receptor for SARS-CoV-2 and SARS-CoV, revealed abundant expressions in canine NC and SP epithelia, with the most intense staining in the middle and bottom cell layers, respectively ( Figure 2 ). In canine T and L epithelia, ACE2 expressions ranged from rare to mild.

Influenza viruses of different subtypes readily infected and replicated in the canine tissue explants ( Figure 3 ). Canine H3N2, being highly contagious in dogs, replicated efficiently in NC, T and L explants, producing peak virus titres as high as 6.6 log TCID50/ml within 72 hpi ( Figure 3A ). Among the seven human/avian IAV subtypes and two IBV isolates, HPAI H5N1, H5N6 and H7N9 and quail H9N2 replicated most efficiently in NC explants. They shared comparable areas under their replication kinetic curves (AUC) from 24 to 72 hpi with that of canine H3N2 ( Figure 3B ). A c c e p t e d M a n u s c r i p t 12 to 5.1 log TCID 50 /ml, which is only around 1 log lower than that of canine H3N2. In L explants, human/avian IAVs, Victoria-like IBV and canine H3N2 showed comparable AUC values with the means of their peak virus titres ranging between 4.5 to 5.7 log TCID 50 /ml.

Meanwhile, Yamagata-like IBV yielded peak virus titres between the undetected level, ≤ 1.5 log TCID 50 /ml, to 3.3 log TCID 50 /ml.

To determine the SA receptor distribution in the canine tissues, we performed lectin histochemistry. SNA binding (specific towards α2,6-linked SA) was abundant in the epithelia of NC, T and bronchioles, but moderate in SP and alveolus ( Figure 4) . The MAAI and MAAII isotypes, which preferentially bind N-linked or core 2 O-linked glycans containing SAα2,3-Galβ1,4GlcNAc and O-linked glycans containing SAα2,3-Galβ1,3GalNAc, respectively [22, 23] , displayed distinct distribution patterns. MAAI binding was abundant in the epithelia of SP and bronchioles, moderate at the level of NC and rare in T and alveolus.

Meanwhile, MAAII binding was abundant in the epithelia of all tissue explants. Note that MAAI and MAAII are also known to bind with high affinity to non-SA glycans containing SO 4 -3-Galβ1,4GlcNAc and SO 4 -3-Galβ, respectively [22, 23] .

We showed in this study the limited permissiveness of canine respiratory and soft palate [30] , avian H5N1 [31] and avian H9N2 [32] have been detected or isolated in dogs shortly after the emergence of canine H3N2. Hence, we were particularly concerned at the fact that HPAI H5 and H7 viruses and the highly prevalent avian H9N2 virus, which had been shown to be a donor of internal genes to highly zoonotic viruses [33] , had fairly high replication efficiencies in all canine tissues tested. Heterosubtypic reassortments may be more likely to occur in canine T and L where most influenza subtypes in this study replicated well providing greater chances of co-infections. Previously, higher genomic diversity was also found among reassortants recovered from the middle and lower respiratory tracts in experimental IAV co-infected swine [34] . NC and SP which are highly exposed the environment have been shown to be important sites of generation and adaptation for transmissible influenza viruses in ferrets [35, 36] . Whether the same applies to dogs remains to be answered. However, we demonstrated that canine NC and SP are among the initial sites of infection and amplification for human and avian influenza viruses. Higher viral loads at these sites could contribute in part to further dissemination through coughing, sneezing, breathing and licking as well as better infection of the deeper tissues. Yet, it has to be noted that the physiological temperature in the canine nose at an ambient temperature of around 20 o C is approximately 34 o C, which is lower than the 37 o C set for canine NC explants in our study. The difference in temperatures may lead to variations in viral replication at this site. Previous studies using the same strains of canine and human seasonal H3N2 and 2009 H1N1 pdm indicated similar to better replication efficiencies at 32-33 o C than 37 o C in primary human nasal and bronchial epithelial cells [16] and MDCK cells [37] , respectively. The effect was the opposite for the replication of quail H9N2 in primary human bronchial epithelial cells [17] and the polymerase activity of HPAI H5N6 in 293T cells [38] .

A c c e p t e d M a n u s c r i p t 15 Although viral shedding of SARS-CoV-2 and SARS-CoV appears to be insufficient to cause efficient dissemination, the possibility of genetic recombination should be aware of given the plenty of chances for spillover during outbreaks and the high prevalence of canine respiratory coronavirus, which belongs to the same beta-CoV genus, in dogs [39] . Co-infection between SARS-CoV-2 and influenza virus in dogs as it happened in humans [40] is another concern.

The ways the two viruses interact and their influence on each other in replication, transmission and pathogenesis is yet to be understood.  current dominant pandemic form.

 higher viral loads in patients.

 higher fatality rate. A c c e p t e d M a n u s c r i p t 

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generous sharing of virus seed stocks. We would also like to express our deepest gratitude to all the dogs whose carcasses were donated for the experiments. May they rest in peace.

A c c e p t e d M a n u s c r i p t