Adaptation of H3N2 canine influenza virus to feline cell culture.

H3N2 canine influenza viruses are prevalent in Asian and North American countries. During circulation of the viruses in dogs, these viruses are occasionally transmitted to cats. If this canine virus causes an epidemic in cats too, sporadic infections may occur in humans because of the close contact between these companion animals and humans, possibly triggering an emergence of mutant viruses with a pandemic potential. In this study, we aimed to gain an insight into the mutations responsible for inter-species transmission of H3N2 virus from dogs to cats. We found that feline CRFK cell-adapted viruses acquired several mutations in multiple genome segments. Among them, HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations individually increased virus growth in CRFK cells. With a combination of these mutations, virus growth further increased not only in CRFK cells but also in other feline fcwf-4 cells. Both HA1-K299R and HA2-T107I mutations increased thermal resistance of the viruses. In addition, HA2-T107I increased the pH requirement for membrane fusion. These findings suggest that the mutations, especially the two HA mutations, identified in this study, might be responsible for adaptation of H3N2 canine influenza viruses in cats.

Introduction

Influenza A viruses are maintained in wild aquatic birds as natural reservoirs [1]. The waterfowl viruses are occasionally transmitted to poultry, causing several diseases. Although these avian viruses are rarely transmitted to mammals, specific subtypes of viruses have become endemic to humans and other mammals, including pigs and horses [2]. Among mammals, dogs and cats were thought to be historically restricted to influenza virus infections [3], although evidence of the infections in these companion animals have been sporadically reported [4-9]. Recently, two subtypes, H3N8 and H3N2, of canine influenza viruses (CIVs) have emerged and are endemic in dogs. H3N8 CIV was first detected among racing greyhounds in Florida, USA, in 2004, and studies revealed that the virus arose as a result of direct transmission of an H3N8 equine virus from a neighboring horse [10]. H3N2 CIV was first isolated in South Korea in 2007 and was revealed to be of avian virus origin [11]. Further studies showed that H3N2 CIV had been already present in southern China and South Korea before 2007 [12,13]. In 2012, the H3N2 CIV was isolated in Thailand [14], indicating that the virus had spread among the canine population in Asian countries. In 2015, H3N2 CIV caused an outbreak in an animal shelter in Chicago, USA, probably by importation of infected dogs from Asia [15]. Since then, H3N2 CIV infection has expanded nationwide in the USA, as has the H3N8 CIV [16]. Multiple subtypes of avian-origin viruses, such as H5N1, H5N2, H6N1, and H9N2, were isolated from dogs with respiratory illness in Asia since 2014 [17-19]. From 2013 to 2015, H1N1 swine origin-reassortant viruses were transmitted to dogs from pigs, and H1N1, H1N2, and H3N2 reassortant viruses of swine and canine H3N2 viruses were further detected in dogs in southern China [20].

Reports have shown that several human and avian influenza viruses can infect domestic cats. Serological surveys suggested that H3N2 human seasonal viruses can be transmitted to cats [21,22] and H1N1pdm2009 human virus managed to infect a cat colony, causing severe clinical diseases with fatalities [23]. The highly pathogenic H5N1 avian influenza virus (HPAIV) naturally infects domestic cats and causes severe illnesses [24]. In 2016, a low pathogenic strain of H7N2 AIV was transmitted into cats in an animal shelter and a veterinarian, who took care of these cats, got infected with the virus transmitted from the infected cats, causing mild respiratory symptoms [25]. In addition, under experimental conditions, domestic cats have been known to be susceptible to other influenza viruses such as H2N2 human, H7N7 seal, and H7N3 avian viruses [3,26,27].

Recently, transmission of H3N2 CIV into cats was also reported in two animal shelters in South Korea, where dogs and cats were accommodated together, resulting in 21.7% and 40% mortality of the cats in each shelter, respectively; the affected cats exhibited severe respiratory signs [28,29]. The transmission of H3N2 CIV into cats was also reported in China [20].

Mutation and reassortment are critical for cross-species transmission of influenza A virus, thereby establishing a new lineage in the new host. For example, H3N8 CIV emerged due to mutations in H3N8 equine virus [10], and H1N1pdm2009 virus emerged due to the reassortment between avian, swine, and human viruses with human-adaptive mutations [30]. Because of a frequent close contact between companion animals and humans, influenza viruses of companion animals pose a threat of being transformed into a pandemic virus by causing mutations or reassortment with human seasonal viruses. Indeed, a reassortant between H3N2 CIV and H1N1pdm virus emerged in a dog [31]. If a CIV adapts to cats and spreads in a cat population, the viruses can have more opportunities to be transmitted to humans, as they infect not only cats but also dogs. However, the molecular basis of H3N2 CIV adaptation to cats has not been elucidated.

In this study, to gain an insight into the mutations responsible for inter-species transmission of H3N2 CIVs from dogs to cats, we generated feline cell-adapted CIVs and analyzed the acquired mutations relevant to the viral adaptation.

Materials and methods

Cells and viruses

Madin–Darby canine kidney (MDCK) cells obtained from the American Type Culture Collection (ATCC; CCL-34) were maintained in minimal-essential medium (MEM) supplemented with 5% newborn calf serum (NCS) and antibiotics. Human embryonic kidney 293T cells from RIKEN BioResource Research Center (RCB2202) and fcwf-4 (Felis catus whole fetus) cells from ATCC (CRL-2787) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Crandell–Rees feline kidney (CRFK) cells from ATCC (CCL-94) were maintained in DMEM supplemented with 5% FBS. All the cells were incubated in 5% CO2 at 37˚C. A/canine/Madison/5/2015 (H3N2) virus, kindly provided by Dr. Kathy Toohey-Kurth (Veterinary Diagnostic Laboratory, School of Veterinary Medicine, University of Wisconsin), was propagated in MDCK cells, and the supernatants containing viruses were collected and stored at -80°C.

Plaque assay

Confluent monolayers of MDCK cells were washed with MEM supplemented with 0.3% bovine serum albumin (MEM/BSA), infected with diluted virus, and incubated for 60 min at 37°C. After the virus inoculum was removed, the cells were washed and overlaid with MEM/BSA containing 1% agarose supplemented with 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Worthington, Lakewood, NJ, USA). The plates were incubated at 37°C for 36 h, and then the cells were stained with crystal violet solution (0.1% crystal violet in 20% methanol).

Adaptation of H3N2 canine influenza virus to CRFK cells

CRFK cells, seeded on three dishes of 3.5 cm diameter, were infected with 100 μL of 1000-fold-diluted stock virus (3.5×107 PFU/mL) and maintained in DMEM supplemented with 0.3% BSA (DMEM/BSA) and 0.3 μg/mL TPCK-trypsin. At 48 h post-infection, the media containing viruses were centrifuged to remove cell debris and supernatants were collected. Next, three new dishes with CRFK cells were infected with viruses in 100 μL of 100-fold-diluted supernatants. Viruses obtained after 10 passages were collected as CRFK-adapted (CA) viruses for subsequent experiments.

Sequence analysis

The RNAs of wild-type and CA viruses were extracted from the supernatants using ISOGEN-LS (Nippon Gene, Tokyo, Japan). Reverse transcription of viral RNA was performed using primers containing the conserved sequences at the 3-prime ends of the viral segments using ReverTra Ace (TOYOBO, Osaka, Japan). Later, polymerase chain reaction (PCR) was conducted using specific primer pairs for each gene segment [32]. PCR products were purified using the Fast Gene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and sequenced directly using specific primers in an automated sequencer (Life Technologies Japan, Applied Biosystems 3130xl, Tokyo, Japan).

Reverse genetics

The cDNAs of eight viral segments obtained from A/canine/Madison/5/2015 or CA viruses were cloned into the genomic RNA expression plasmid, pHH21 [33]. Escherichia coli DH5α cells transformed with the cDNA-inserted plasmids were incubated at 37°C on LB agar plates supplemented with ampicillin. All the plasmids were sequenced to ensure that unexpected mutations were not present. We also generated viral polymerase and NP expression plasmids by inserting the coding sequences of A/WSN/33 segments into pCAGGS. Wild-type and mutant viruses were generated by reverse genetics as described previously [33]. Briefly, viral RNA expression, viral protein PB2-, PB1-, PA-, and NP-expression plasmids were mixed with a transfection reagent (TransIT-293; Mirus Bio, Madison, WI, USA), incubated at 21°C for 15 min, and the mixtures were added to 293T cells. Transfected cells were incubated in Opti-MEM (Life Technologies/GIBCO, Grand Island, NY, USA) for 48 h. Supernatants containing the infectious viruses were harvested and propagated in MDCK cells maintained in MEM/BSA supplemented with 1 μg/mL TPCK-trypsin for 48 h, and then the virus-containing supernatants were aliquoted and stored at -80°C.

Virus growth kinetics in cultured cells

MDCK cells were infected with viruses at a multiplicity of infection (moi) of 0.001 and maintained in MEM/BSA with 1 μg/mL TPCK-trypsin. CRFK and fcwf-4 cells were infected with viruses at an moi of 0.01 and maintained in DMEM/BSA with 0.3 μg/mL and 0.4 μg/mL TPCK-trypsin, respectively. Supernatants were collected every 12 h and virus titers were measured using plaque assay.

Fusion assay

Cell fusion assay was performed as previously described [34]. The cDNA of HA of wild-type virus or mutant viruses bearing HA1-K299R or HA2-T107I mutations were cloned into the protein expression plasmid, pCAGGS. All the plasmids were sequenced to ensure that unintended mutations were not present. MDCK cells were co-transfected with wild-type HA or mutant HA (HA1-K299R or HA2-T107I) expression plasmids, as well as with a fluorescent protein (Venus) expression plasmid (pcDNA3.1-Venus) by using polyethylenimine (PEI; Polysciences, Inc., Warrington, PA, USA). After transfection, the cells were incubated at 37°C for 24 h. The cells were then washed twice with Mg2+ and Ca2+ containing phosphate-buffered saline (PBS+) and treated with 5 μg/mL of TPCK-trypsin for 5 min at 37°C. Later, the trypsin was inactivated by washing with 4% NCS in PBS+. To induce cell fusion, the cells were treated with pH-adjusted PBS+ by citric acid for 1 min and incubated in growth medium at 37°C for 60 min. The fused cells were observed under a fluorescence microscope (Axio Vert.A1: ZEISS, Oberkochen, Germany).

Thermostability assay

Thermostability assay was conducted as previously described [35]. Briefly, wild-type virus or mutant viruses bearing HA1-K299R or HA2-T107I were diluted to 4×106 PFU/mL and divided into six aliquots of 180 μL each. All the aliquots were heated at 42°C for 0, 15, 30, 60, 90, and 120 min, and then placed on ice immediately. Infectivities of the heated viruses were determined by plaque assay.

Biosafety statement

All experiments with infectious viruses were approved by the University of Tokyo and conducted in BSL2 laboratories.

Results

Adaptation of an H3N2 CIV to CRFK cells

We generated feline cell-adapted H3N2 CIVs. A CIV strain was serially passaged 10 times in CRFK cells at 2-day intervals. After the 4th passage of these viruses, cytopathic effects were clearly visible, and rapidly expanded in the whole area of cells, leading to detachment of infected cells from the surface of the plate at the 9th passage. Subsequently, we used three lines of viruses from the 10th passage onwards as CRFK-adapted CIVs for further analyses, referred as Mad-CA1, -CA2, and -CA3. We compared the growth kinetics of wild-type and Mad-CA viruses in CRFK cells (Fig 1). Mad-CA viruses showed significantly higher titers than that of wild-type virus at each time point, indicating that these viruses were adapted to feline cells.

Fig 1

Growth kinetics of CRFK cell-adapted viruses in CRFK cells.

CRFK cells were infected with CRFK cell-adapted viruses (Mad-CA1, -CA2, or -CA3) or wild-type (WT) virus at an moi of 0.01, supernatants were collected every 12 h, and virus titers of supernatants were measured. Error bars represent standard deviation (SD) of the means from three independent experiments. The statistical differences in the growth between the CRFK cell-adapted and wild-type viruses were assessed by two-tailed Student’s t-test (**p<0.01).

Interestingly, we were unable to obtain any CIVs that adapted to human-derived A549 or monkey-derived Vero cells (data not shown).

Sequencing of CRFK cell-adapted CIVs

To identify the amino acid substitutions in the Mad-CA viruses, their complete genomes were sequenced and compared to those of the wild-type virus (Table 1). There were several nonsynonymous mutations in HA, NP, NA, and M segments. Notably, no mutation was found in the other segments including polymerase genes, which typically undergo several mutations during interspecies transmission of the virus [36]. Among them, we focused on mutations of envelope proteins such as HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations, three of which were observed in two or more Mad-CA viruses.

Table 1

Amino acids at the positions where mutations were observed in CRFK-adapted CIVs.

Virus	Segment
	HA		NP		NA	M
	HA1-299	HA2-107	289	309	35	M1-15	M2-41
Wild-type	K	T	Y	N	L	I	W
Mad-CA1	K	I	C	N	R	I	C
Mad-CA2	R	T	Y	N	R	T	C
Mad-CA3	R	T	Y	S	R	I	C

Mutations responsible for CIV adaptation to CRFK cells

To characterize the mutations observed in the Mad-CA viruses, we generated viruses possessing single mutation (HA1-K299R, HA2-T107I, NA-L35R, or M2-W41C), viruses possessing double mutations (HA1-K299R/NA-L35R, or HA1-K299R/M2-W41C), and a virus possessing triple mutations (HA1-K299R/NA-L35R/M2-W41C), in addition to the wild-type virus by reverse genetics, referred as rHA1-K299R, rHA2-T107I, rNA-L35R, rM2-W41C, rHA1-K299R/NA-L35R, rHA1-K299R/M2-W41C, rHA1-K299R/NA-L35R/M2-W41C, and rWT, respectively. Next, CRFK cells were infected with each recombinant virus, and the virus titers were measured. The plaque sizes were similar in all the eight generated viruses in MDCK cells. The titers of all the four single amino acid mutants were significantly higher than that of rWT virus at most time points. Both the HA mutants, rHA1-K299R and rHA2-T107I, enhanced virus growth at higher levels than that by rNA-L35R and rM2-W41C (Fig 2A). In addition, all the double or triple mutants grew better than single amino acid mutants, indicating synergistic effects of the mutations (Fig 2B). These data suggest that each mutation has an independent mechanism of virus growth in CRFK cells and is involved in the CIV adaptation to CRFK cells.

Fig 2

Growth kinetics of the mutants generated by reverse genetics in CRFK cells.

CRFK cells were infected with recombinant viruses possessing (A) single mutation (rHA1-K299R, rHA2-T107I, rNA-L35R, or rM2-W41C), (B) multiple mutations (rHA1-K299R/NA-L35R, rHA1-K299R/M2-W41C, or rHA1-K299R/NA-L35R/M2-W41C), or recombinant wild-type (rWT) virus at an moi of 0.01. The virus titers were measured at each time point. Error bars represent SD of the means from three independent experiments. The statistical differences in the growth between the mutant viruses and rWT were assessed by two-tailed Student’s t-test (*p<0.05, **p<0.01).

Fusion activity of the HA mutants

Amino acid positions, 299 in HA1 and 107 in HA2, were located near and inside the stalk region of the HA. Therefore, we suspected that these mutations would affect membrane fusion activity of the HA. We conducted a cell fusion assay by co-transfecting wild-type or mutant HA expression plasmids and a Venus expression plasmid into MDCK cells. At 24 h post transfection, cells were exposed to several low-pH buffers, and membrane fusion was observed (Fig 3). We did not find any fused cells in wild-type and mutant HA expressing cells at a pH range of more than 5.8. In contrast, at pH 5.0, 5.2, and 5.4, fused cells were observed in both the wild-type and mutant HA expressing cells. At pH 5.6, fused cells were observed only in mutant T107I HA expressing cells. These data suggest that HA2-T107I mutation widens the optimal pH range for virus membrane fusion in MDCK cells.

Fig 3

pH range of syncytium formation of the mutant HAs in MDCK cells.

MDCK cells were co-transfected with a mutant (HA1-K299R or HA2-T107I) or wild-type (HA-WT) HA expression plasmid and a Venus expression plasmid. After treatment with the indicated pH buffers, the cells were observed under a fluorescence microscope. The red lines indicate the boundaries of pH at which cell fusion occurred. The assay was conducted three times independently and the representative data are shown.

Thermostability of the HA mutants

To investigate a mechanism why HA1-K299R and HA2-T107I mutations increased virus growth in CRFK cells, we analyzed the thermostability of the HA mutant viruses. Each virus was diluted, incubated at 42°C for 0, 15, 30, 60, 90, and 120 min, and the reduction of virus titers was measured. We observed that the reduction rates in the titers were not as marked in the HA mutants, compared to those in rWT virus (Fig 4). These data suggest that both the HA1-K299R and HA2-T107I mutations confer thermostability to the CIV, leading to its enhanced growth in CRFK cells.

Fig 4

Thermal stability of the mutant viruses.

The mutant (rHA1-K299R and rHA1-T107I) and wild-type (rWT) viruses were diluted to 4×106 PFU/mL and aliquoted. After the aliquots were heated at 42°C for 0, 15, 30, 60, 90, or 120 min, virus titers were measured. Error bars represent SD of the means from three independent experiments. Statistical differences in the titers between the mutant viruses and wild-type virus (rWT) were assessed by two-tailed Student’s t-test (*p<0.05, **p<0.01).

Growth of the mutants in MDCK cells

To examine whether the mutations affect the virus growth in canine cells, we assessed growth kinetics of the mutants in MDCK cells. Interestingly, the viruses possessing HA1-K299R or HA2-T107I mutations grew better than rWT at some time points, albeit small differences were observed in their titers. In contrast, the rNA-L35R and rM-W41C viruses exhibited similar growth properties to rWT (Fig 5A). Growth kinetics with a combination of the mutations confirmed these observations (Fig 5B). These data demonstrated that mutations required for feline cell-adaptation of the CIV have little effect on the virus growth in canine cells. Notably, all of the mutants were unable to replicate in human-derived A549 or monkey-derived Vero cells, similar to rWT (data not shown).

Fig 5

Growth kinetics of the mutants in MDCK cells.

MDCK cells were infected with recombinant viruses possessing (A) single mutation (rHA1-K299R, rHA2-T107I, rNA-L35R, or rM2-W41C), (B) multiple mutations (rHA1-K299R/NA-L35R, rHA1-K299R/M2-W41C, or rHA1-K299R/NA-L35R/M2-W41C), or recombinant wild-type (rWT) virus at an moi of 0.001. The virus titers were measured at each time point. Error bars represent SD of the means from three independent experiments. Statistical differences in the growth between the mutant viruses and rWT were assessed by two-tailed Student’s t-test (*p<0.05, **p<0.01).

Growth of the mutants in fcwf-4 cells

We examined growth of the mutants in other type of feline cells ‒ macrophage-like fcwf-4 cells. All the viruses possessing HA1-K299R or HA2-T107I mutations grew better than rWT as did in epithelial-type CRFK cells. In contrast, growth kinetics of the viruses possessing single NA-L35R or M-W41C mutations were similar to those of rWT (Fig 6A). However, NA-L35R mutation enhanced the viral growth in combination with HA1-K299R mutation in a synergistic manner (Fig 6B). These data indicated that HA1-K299R or HA2-T107I mutations were primary determinants for feline cell-adaptation of the CIV.

Fig 6

Growth kinetics of the mutants in fcwf-4 cells.

Fcwf-4 cells were infected with recombinant viruses possessing (A) single mutation (rHA1-K299R, rHA2-T107I, rNA-L35R, or rM2-W41C), (B) multiple mutations (rHA1-K299R/NA-L35R, rHA1-K299R/M2-W41C, or rHA1-K299R/NA-L35R/M2-W41C), or recombinant wild-type (rWT) virus at an moi of 0.01. The virus titers were measured at each time point. Error bars represent SD of the means from three independent experiments: only upward error bars are represented. The statistical differences in the growth between the mutant viruses and rWT were assessed by two-tailed Student’s t-test (*p<0.05, **p<0.01).

Discussion

The recent spread of CIVs in some Asian countries and the USA raises a public health concern that the virus could be transmitted to humans directly or through intermediate hosts, causing a new pandemic [20]. Cats are probable candidates that could act as intermediate hosts, since they have a close relationship to humans as companion animals. Previous reports showing H3N2 CIV transmission to cats may accentuate this idea [28,29]. If a CIV adapts to cats and spreads in the cat population, the viruses have more opportunities to be transmitted to humans, as they can infect not only cats but also dogs. However, the molecular basis of H3N2 CIV adaptation to cats has not been elucidated. Here, we generated feline cell-adapted CIVs and revealed that both HA1-K299R and HA2-T107I mutations enhanced virus growth in feline cell lines, by altering the optimal pH for viral membrane fusion and/or promoting thermostability of the virus. Previous studies have shown that such alterations of HAs may play a role in the adaptation of H1N1, H5N2, and H9N2 viruses to different host cells and animals [35,37-39]. In addition, we found that NA-L35R and M-W41C mutations could improve CIV growth in feline cell lines, albeit less efficiently, compared to HA mutations. These findings suggest that HA1-K299R, HA2-T107I, NA-L35R, and M-W41C mutations might be responsible for CIV adaptation to cats, although studies need to be conducted by experimentally infecting the feline cell-adapted CIVs into cats.

Two H3N2 CIVs were isolated from cats at animal shelters in Korea in 2010, revealing direct transmission of the virus from dogs to cats [28,29]. In one study, CIVs were transmitted from dogs in the same shelter, where the virus was circulating among the dogs [28]. In this paper, 18 amino acid differences were found in genomes between the dog and cat isolates, all of which were not observed in our study. Among them, two HA mutations, HA1-S107P and -S116G (H3 numbering), were found in cat isolates, implicating that these mutations might be responsible for CIV adaptation to cats. The amino acid positions at these mutations were located at bottom of head region of the HA. Interestingly, these locations are near the position 299 in HA1-K299R mutation found in our study (Fig 7A). An undefined mechanism related to these HA1 mutations may be associated with CIV adaptation to cats.

Fig 7

3D structure of H3-HA protein.

The 3D structure of HA of A/Aichi/2/1968 (H3N2) (PDB: 1HGG) is shown. (A) Amino acid positions, HA1-299 and HA2-107, at which mutations were observed in the CRFK-adapted CIVs, are colored orange or cyan, respectively. Amino acid positions, 107 and 116, in HA1, at which substitutions were observed in the feline isolates of CIV [28], are colored green. (B) Close-up view of the area surrounding HA1-299 is shown. A polar contact is formed between HA1-K299 and HA2-E69. (C) Close-up view of the area surrounding HA2-107 is shown. A polar contact is formed between HA2-T107 and HA2-K51.

Although a previous study showed that K299G or K299E mutations in HA1 of X-31 strain (H3 subtype) increased the optimal pH for conformational change into membrane fusion [40], CIV HA1-K299R mutation observed in our study did not affect the optimal pH of the membrane fusion (Fig 3). Instead, this mutation lead to thermal resistance of the mutant virus (Fig 4). Lysine at position 299 of H3 HA forms a polar contact with HA2-69E [41] (Fig 7B), and this contact strength may have increased by lysine to arginine substitution, because arginine is more negatively charged than lysine. Such a change in polar contact strength could have increased the stability of the mutant virus. On the contrary, HA2-T107I mutation increased both the optimal pH of membrane fusion as well as thermal resistance of virus. Previous studies showed that polar contacts, which are formed between amino acids around HA2 position 107 and HA2 positions 39–55 in α-helix structure, markedly affected the optimal pH for membrane fusion [42-44]. In our study, HA2-T107I mutation may have lost a polar contact with HA2-51K (Fig 7C), thereby increasing the optimal pH for membrane fusion. Additional analyses are required to elucidate the mechanisms of changes in thermal stability by mutations.

Amino acids at position 31–35 of NA transmembrane domain are involved in its raft association [45]. Therefore, NA-L35R mutation found in the mutants may affect the raft association of NA, resulting in a change in the amount of this protein in the virions. The change in NA content in the virions may have altered the HA-NA functional balance, leading to high growth of the mutant in feline cells.

An ion channel M2 protein located on virus envelope is activated under acidic pH condition of endosomes [46]. A histidine residue at position 37 and a tryptophan residue at position 41 have cation-π interaction (Fig 8) [47]. This interaction is important for the proton-selective ion channel activity of M2, and M2-41W residue, which plays a role as a gate-keeper to prevent spilling out of protons from inside the acidic virions [48,49]. M2-W41C mutation caused high membrane conductance under high pH condition of an external virion [49], suggesting that the M2-W41C mutant could allow protons to flow into the virion even at a higher pH of the endosomes. Together with HA2-T107I mutation that raises the pH condition for membrane fusion, CIVs may have been adapted to endosomal pH environment in the feline cells by HA as well as M2 mutations.

Fig 8

3D structure of M2 protein.

The 3D structure of M2 of A/Udorn/1972 (H3N2) (PDB: 2RLF) is shown. H37 and W41, which possess cation-π interaction, are colored green and red, respectively.

In conclusion, we identified multiple mutations required for adaptation of H3N2 CIV to feline cells. Although a study with experimental infection of feline cell-adapted viruses in cats is required, the mutations found in these viruses may contribute to understand the mechanism on cross-species transmission of CIVs and risk assessment for pandemic preparedness.

2 Aug 2019

PONE-D-19-18114

Adaptation of H3N2 canine influenza virus to feline cell culture

PLOS ONE

Dear Prof. Horimoto,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Sep 16 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Kensuke Hirasawa, PhD

Academic Editor

PLOS ONE

Journal Requirements:

1.  When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes adaptation of an influenza A virus strain that was isolated from a dog to a cat derived cell line. The authors analyzed genetic changes and their effects on the virus phenotypes. They initially identified the mutations after a serial passage, then introduced the found mutations individually or in combination into the genetic background of the parental dog virus genome by reverse genetics. The phenotypes of these recombinant viruses were tested in vitro. The study is original, methodology was sound and the results were properly analyzed and presented. However, I would ask the authors several questions regarding the motivation and the design of this study, and the interpretation of the results:

1. The authors tried to identify the required mutation for a dog influenza A virus to adapt to cat. Why the authors thought cat-adapted influenza A virus would be more dangerous than dog virus to human (line 73-74 & 311-312)? The authors stated that “cats are probable candidates that could act as intermediate hosts, …” (line 308-309). Aren’t dogs or other mammalian species, as well?

2. The authors passed dog derived influenza A virus in CRFK cells to adapt the virus to cat cells. Are these viruses really better fit to cat, though they stated “… CIV adaptation to cats.” (line 319)? Good virus replication in a host is essential for the virus spread to another host, however, is there any evidence that supports parallel relationship between the replication efficiency and the transmission efficiency?

3. The authors presented evidence that HA1-K299R and HA2-T107I mutations enhance virus growth in cat derived cells, alter the optimal (threshold?) pH for membrane fusion and thermostability. However, these do not necessarily mean the latter alterations were the cause for the former enhanced virus growth in cat cells. Why the altered optimal pH for fusion and thermostability are preferable for the virus to grow in cat (but not dog) cells (line 315)? Isn’t higher thermostability preferable for the virus replication in dog cells in vitro, as well? Is there any evidence or literature that support the authors’ speculation?

4. The authors compared a cat isolate in a literature to their own CRFK adapted virus (P. 15, last paragraph). When an influenza A virus adapted to another host species NS1 and PB2 were mutated in previous literatures (ref. 26 and J. Virol. 84, 10606-). Is there any possible reason why neither gene did not mutate in your experiment?

5. The authors stated “CIVs may have been adapted to endosomal pH environment in feline cells …” (line 369). How the endosomal environment is different between in CRFK and in MDCK? Do you have any information? This would also help answer the question 3 above.

Minor points

• Line 51-64: This paragraph should be better organized. Transmissions cannot be isolated (line 59-60).

• Line 234: Figures should appear in the text in numerical order; Fig. 7A should not appear before Fig. 3.

• Fig. 3: The pictures need better focus.

Reviewer #2: This manuscript described that canine influenza virus H3N2 was atapted to feline cells and the mechanism of adaptation was analyzed. The authors discussed that two mutations are responseible for adaptation of H3N2 canine influenza virus in cats and the mechanisms were thermal resistance and pH requirement. However, my major comments should be addressed.

Major comments

These mutations might occur after adaptation to in vitro cell culture, but not only to feline cell culture. It should be denied that these mutations occurred after adaptation to cell culture. Canine influenza virus H3N2 should be adapted to the other mammalian cell lines.

These recombinant viruses should be analyzed in the other cells line, but not only in MDCK, CRFK and fewf-4 cells.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Sep 2019

Responses to Reviewer #1:

“1. The authors tried to identify the required mutation for a dog influenza A virus to adapt to cat. Why the authors thought cat-adapted influenza A virus would be more dangerous than dog virus to human (line 73-74 & 311-312)? The authors stated that “cats are probable candidates that could act as intermediate hosts, …” (line 308-309). Aren’t dogs or other mammalian species, as well?”

We apologize for the unclear statement. We intended to argue that both companion animals, dogs and cats, are potential candidates that can act as intermediate hosts, due to their close contact to humans. To clarify this point, we altered the sentence in the revised manuscript (lines 75/319-320) as follows:

“If a CIV ……, the viruses …., as they infect not only cats but also dogs.’’

“2. The authors passed dog derived influenza A virus in CRFK cells to adapt the virus to cat cells. Are these viruses really better fit to cat, though they stated “… CIV adaptation to cats.” (line 319)? Good virus replication in a host is essential for the virus spread to another host, however, is there any evidence that supports parallel relationship between the replication efficiency and the transmission efficiency?”

We agree with this comment. However, to make this point clear, experimental infection and transmission studies with feline cell-adapted viruses in cats are required. We are planning to do these experiments in cats as a next step. Therefore, we rephrased the sentence in the revised manuscript (line 329-330) as follows:

“These findings……mutations may be responsible for CIV adaptation to cats, although studies need to be conducted by experimentally infecting the feline cell-adapted CIVs into cats.”.

“3. The authors presented evidence that HA1-K299R and HA2-T107I mutations enhance virus growth in cat derived cells, alter the optimal (threshold?) pH for membrane fusion and thermostability. However, these do not necessarily mean the latter alterations were the cause for the former enhanced virus growth in cat cells. Why the altered optimal pH for fusion and thermostability are preferable for the virus to grow in cat (but not dog) cells (line 315)? Isn’t higher thermostability preferable for the virus replication in dog cells in vitro, as well? Is there any evidence or literature that support the authors’ speculation?”

We agree with this comment. However, we have no direct evidence to support our speculation. Therefore, we added a sentence with references reporting that the changes in optimal HA for membrane fusion or thermostability could be related to replication and transmission of the viruses to other host cells and animals in the revised manuscript (lines 324-325) as follows:

“Previous studies have shown that such alterations of HAs may play a role in the adaptation of H1N1, H5N2, and H9N2 viruses to different host cells and animals [35,37-39].”

We referred to these papers in the revised manuscript.

37. Sang et al., Adaptation of H9N2 AIV in guinea pigs enables efficient transmission by direct contact and inefficient transmission by respiratory droplets. Sci Rep. 2015;5: 15928.

38. Klein et al., Stability of the influenza virus hemagglutinin protein correlates with evolutionary dynamics. mSphere. 2018;3: e00554-17.

39. Singanayagam et al., Influenza virus with increased pH of hemagglutinin activation has improved replication in cell culture but at the cost of infectivity in human airway epithelium. J Virol. 2019;93: e00058-19.

“4. The authors compared a cat isolate in a literature to their own CRFK adapted virus (P. 15, last paragraph). When an influenza A virus adapted to another host species NS1 and PB2 were mutated in previous literatures (ref. 26 and J. Virol. 84, 10606-). Is there any possible reason why neither gene did not mutate in your experiment?”

As the reviewer has rightly pointed out, several mutations in the internal genes such as IFN-antagonized NS1 and polymerase subunit PB2 have been frequently observed, especially during in vivo adaptation of avian viruses to mammalian hosts. Contrastingly, our mutants were obtained through in vitro cell culture passages of a mammalian (dog) virus in mammalian (cat) cells. Therefore, we speculate that the difference in the methods adopted for viral adaptation between the previous studies and our study could be a possible reason why these internal genes did not mutate in our mutants. We have added a sentence addressing this point in the revised manuscript (lines 200-202).

“5. The authors stated “CIVs may have been adapted to endosomal pH environment in feline cells …” (line 369). How the endosomal environment is different between in CRFK and in MDCK? Do you have any information? This would also help answer the question 3 above.”

We could not obtain any information on the difference in the endosomal pH environments between CRFK and MDCK cells. Therefore, we just made a mention of this issue in the manuscript (lines 380-381) as follows:

“CIVs may have been adapted to endosomal pH environment in feline cells by HA as well as M2 mutations.”

Minor points

“Line 51-64: This paragraph should be better organized. Transmissions cannot be isolated (line 59-60).”

We rearranged this paragraph in the revised manuscript (lines 51-65) as follows:

“Reports have shown that several human and avian influenza viruses can infect domestic cats. Serological surveys suggested that H3N2 human seasonal viruses have been transmitted to cats [21,22] and H1N1pdm2009 human virus managed to infect a cat colony, causing severe clinical diseases with fatalities [23]. The highly pathogenic H5N1 avian influenza virus (HPAIV) naturally infects domestic cats and causes severe illnesses [24]. In 2016, a low pathogenic strain of H7N2 AIV was transmitted to cats in an animal shelter and a veterinarian, who took care of these cats, got infected with the virus transmitted from the infected cats, causing mild respiratory symptoms [25]. In addition, under experimental conditions, domestic cats have been known to be susceptible to other influenza viruses such as H2N2 human, H7N7 seal, and H7N3 avian viruses [3,26,27].

Recently, transmission of H3N2 CIV into cats was also reported in two animal shelters in South Korea, where dogs and cats were accommodated together, resulting in 21.7% and 40% mortality of the cats in each shelter, respectively; the affected cats exhibited severe respiratory signs [28, 29]. The transmission of H3N2 CIV into cats was also reported in China [20].”

“Line 234: Figures should appear in the text in numerical order; Fig. 7A should not appear before Fig. 3.”

We deleted “Fig. 7A” in the revised manuscript (line 239), as it was not necessary in the paper.

“Fig. 3: The pictures need better focus.”

We apologize for this. However, we were unable to replace the pictures as we have used the best images we had in Fig. 3.

Responses to Reviewer #2:

“These mutations might occur after adaptation to in vitro cell culture, but not only to feline cell culture. It should be denied that these mutations occurred after adaptation to cell culture. Canine influenza virus H3N2 should be adapted to the other mammalian cell lines.”

We were unable to obtain any CIVs adapted to human-derived A549 or monkey-derived Vero cells. In fact, even wild-type CIVs hardly replicated in these cells. We have added this observation in the revised manuscript (lines 186-187).

“These recombinant viruses should be analyzed in the other cells line, but not only in MDCK, CRFK and fewf-4 cells.”

Feline CRFK cell-adapted CIVs were unable to replicate in human A549 and monkey Vero cells as was the parent virus. We have added this observation in the revised manuscript (lines 281-283).

Submitted filename: PlosOne_dog_H3N2_Response_to_Reviewer.edi.docx

Click here for additional data file.

24 Sep 2019

Adaptation of H3N2 canine influenza virus to feline cell culture

PONE-D-19-18114R1

Dear Dr. Horimoto,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Kensuke Hirasawa, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: This revised manuscript was modified according to our comments. Now I agree this manuscript is acceptable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

2 Oct 2019

PONE-D-19-18114R1

Adaptation of H3N2 canine influenza virus to feline cell culture

Dear Dr. Horimoto:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kensuke Hirasawa

Academic Editor

PLOS ONE