Evolution of Canine and Equine Influenza (H3N8)

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Virology 408 (2010) 71–79

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Virology

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Evolution of canine and equine inﬂuenza (H3N8) viruses co-circulating between 2005 and 2008

Pierre Rivailler a, Ijeoma A. Perry a, Yunho Jang a, C. Todd Davis a, Li-Mei Chen a, Edward J. Dubovi b,⁎, Ruben O. Donis a,⁎

a Inﬂuenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA b Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

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Article history: Influenza virus, subtype H3N8, was transmitted from horses to greyhound dogs in 2004 and subsequently Received 23 June 2010 spread to pet dog populations. The co-circulation of H3N8 viruses in dogs and horses makes bi-directional Returned to author for revision 20 July 2010 virus transmission between these animal species possible. To understand the dynamics of viral transmission, Accepted 23 August 2010 we performed virologic surveillance in dogs and horses between 2005 and 2008 in the United States. The Available online 29 September 2010 genomes of influenza A H3N8 viruses isolated from 36 dogs and horses were sequenced to determine their origin and evolution. Phylogenetic analyses revealed that H3N8 influenza viruses from horses and dogs were Keywords: fl Canine influenza virus monophyletic and distinct. There was no evidence of canine in uenza virus infection in horses with Equine influenza virus respiratory disease or new introductions of equine influenza viruses into dogs in the United States. Analysis Host range of a limited number of equine influenza viruses suggested substantial separation in the transmission of Viral evolution viruses causing clinically apparent influenza in dogs and horses. H3N8 Published by Elsevier Inc.

Introduction (Yamanaka et al., 2009). Molecular phylogenies of influenza viruses isolated from these two species since 2004 can help us understand their Equine influenza virus (EIV) subtype H3N8, emerged in Miami transmission dynamics and evolution in the natural environment. To (Florida, USA) in 1963 (Waddell et al., 1963) and subsequently spread to this end, we have analyzed the genomic sequences of 36 viruses isolated equine populations around the world (Barbic et al., 2009; Burrows et al., from dogs and horses between 2005 and 2008 during passive virologic 1981; Damiani et al., 2008; Ito et al., 2008; Newton et al., 2006; Thomson surveillance in four major geographic regions of the United States. et al., 1977; van Maanen and Cullinane, 2002). The global spread of this Phylogenetic analysis of full genomes from CIV isolated since 2005 virus was temporally correlated with a sustained decline in the indicated that these viruses have evolved as a monophyletic group prevalence of the previously dominant H7N7 subtype, which is now rooted in equine influenza genes from earlier times. Similarly, analysis of considered extinct (Webster, 1993). Early studies indicated that equine EIV isolated in the same period (2005–2008) indicated that all their H3N8 viruses did not cause acute respiratory disease in other species, genes belong exclusively to the equine Florida clade 1 (reviewed in including man (Kasel et al., 1965). However, in 2004, an H3N8 virus of (Bryant et al., 2009). These evolutionary patterns indicate that the equine origin caused outbreaks of respiratory disease in dogs and spread interspecies transmission of equine influenza into dogs in 2003–2004 among several urban canine populations of the United States (Crawford led to the emergence of a distinct influenza virus clade that causes a et al., 2005; Payungporn et al., 2008). Subsequent co-circulation of H3N8 transmissible respiratory disease in dogs. Conversely, despite the limited influenza viruses in horses and dogs created the opportunity for further number of EIV analyzed, these findings suggest that influenza-like bi-directional interspecies transmission of influenza viruses between respiratory disease in horses associated with H3N8 viruses is predom- these animal species (Crawford et al., 2005; Parrish and Kawaoka, 2005; inantly caused by equine virus lineages. These studies expand our Payungporn et al., 2008). Experimentally, canine influenza viruses (CIV) understanding of influenza virus ecology in mammals and provide a can replicate in the airway of horses (Yamanaka et al., 2010) whereas foundation for further analysis of host range and virulence determinants. horses infected with EIV can transmit the infection to contact dogs Results

⁎ Corresponding authors. E.J. Dubovi is to be contacted at Animal Health Diagnostic Isolation of subtype H3N8 influenza viruses from dogs and horses with Center, Virology Section, College of Veterinary Medicine at Cornell, Ithaca, NY 14853, respiratory disease USA. Fax: +1 607 253 3943. R.O. Donis, Molecular Virology and Vaccines Branch, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Road-Mail Stop G-16, Atlanta, GA 30333, USA. Fax: +1 404 639 2350. CIV isolates were obtained in both MDCK cells and in embryo- E-mail addresses: [email protected] (E.J. Dubovi), [email protected] (R.O. Donis). nated eggs from samples that were determined to contain influenza

0042-6822/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.virol.2010.08.022 72 P. Rivailler et al. / Virology 408 (2010) 71–79 virus by RT-PCR. In not all instances were both isolation procedures Evolution of canine and equine influenza viruses from 2005 to 2008 successful and neither procedure was better than the other in all cases. This was equally true for influenza virus isolates from equine To investigate the transmission dynamics of H3N8 influenza samples. Overall, isolates were obtained from RT-PCR positive viruses in horses and dogs after the emergence of canine influenza samples with Ct values less than 35 in more than 75% of the cases, virus (CIV) in 2004, we characterized influenza A viruses isolated from and for CT values between 35 and 40, the isolation rate was these species in the United States between 2005 and 2008 (Table 1). approximately 50%. Most clinical samples submitted were swabs and Nucleotide sequence analysis of 36 influenza A viruses that were isolations from swabs simply moistened with saline were easily isolated from dogs and horses revealed the absence of nucleotide obtained. In egg inoculations, samples had to be blind passed at least deletions or insertions in any of the eight viral gene segments. The once before considering the sample as negative due to the relatively phylogenetic relationships of the eight genes of viruses isolated from poor growth of mammalian H3N8 subtype viruses in eggs. Five dogs and horses indicated that they evolved into two distinct clades in specimens yielded isolates from both MDCK and egg inoculations. each of these animal hosts (Figs. 1 and 2, red and yellow shading) Approximately 50% of the isolations that were negative on MDCK diverging from ancestor viruses circulating around 2002–2003. cells were positive with egg isolation. Six of the canine influenza genes (HA, NA, PB2, PB1, PA, and NP) Multiple isolates were obtained from some outbreaks and formed distinct clades statistically supported by bootstrap values representative samples were subjected to sequencing. The 29 canine exceeding 90% (Figs. 1 and 2, red shading) whereas the M and NS influenza A viruses shown in Table 1 were isolated from specimens genes were less divergent, with bootstrap values of 36% and 66%, collected from 6 states representing 3 major geographic regions of the respectively (Figs. 2E and F, red shading). This was expected because United States. The largest number of isolates (13) originated from the M and NS encode for essential viral proteins in different but state of New York. Only 4 isolates originated from racing dogs, the overlapping reading frames, greatly reducing the probability of remaining isolates were from specimens collected from pet dogs. synonymous mutations in the dually coding region and thus Influenza A isolates were obtained from dogs ranging from 4 months constraining divergence (Ito et al., 1991; Kawaoka et al., 1998). to 11 years of age. The specimens from which seven equine viruses However, a subset of more divergent M and NS genes evolved with were isolated were collected from 5 different states representing all 4 bootstrap support above 90% (Figs. 2E and F). major geographic regions of the US: Northeast (New York), Midwest The phylogenetic trees revealed that all genes of A/canine/ (Ohio), South-Central (Virginia) and South-Western (Texas) States Florida/242/03 and A/canine/Florida/43/04 are closely related to (Table 1). contemporary equine viral genes as evidenced by their position on the

Table 1 Inﬂuenza viruses isolated from dogs and horses between 2005 and 2008, USA.

Virus isolate a Host

Strain name Date of collection Passage Breed Age Sex Housing

A/canine/NewYork/115809/2005 30-Sep-05 E2 Labrador 10 yr M Veterinary hospital A/canine/NewYork/4986-2/2006 16-Jan-06 C4 Fox terrier 18 m MN Animal shelter A/canine/NewYork/5183-6/2006 16-Jan-06 C2; E1 Pit bull 12 m M Animal shelter A/canine/Colorado/8880/2006 25-Jan-06 C2 Deerhound 10 m MN Stray from Humane Society A/canine/Colorado/17864/2006 17-Feb-06 C2; E1 Terrier mix 6 m SF Adopted from rescue A/canine/Colorado/30604/2006 20-Mar-06 C2 Labrador 11 yr MN Boarded at positive CIV kennel A/canine/Florida/61156-2/2006 15-May-06 C2; E2 NA NA NA Animal shelter A/canine/California/70645-4/2006 6-Jun-06 E2 Golden retriever 2 yr M Training kennel A/canine/Florida/78592-2/2006b 20-Jun-06 E1 Greyhound NA NA Racing A/canine/Florida/78592-6/2006b 20-Jun-06 C2 Greyhound NA NA Racing A/canine/Florida/78592-7/2006b 20-Jun-06 E2 Greyhound NA NA Racing A/canine/Florida/89911-2/2006 20-Jul-06 E1 Labrador NA NA Shelter dog A/canine/NewYork/100525-1/2006 16-Aug-06 C1 NA NA NA Animal shelter A/canine/NewYork/100528-1/2006 16-Aug-06 C1 NA NA NA Animal shelter A/canine/NewYork/100528-5/2006 16-Aug-06 C1 NA NA NA Animal shelter A/canine/NewYork/100528-6/2006 16-Aug-06 C1 NA NA NA Animal shelter A/canine/Kentucky/118778/2006 20-Oct-06 E1 HoundX 4 m F Humane Society A/canine/NewYork/147926-3/2006 18-Dec-06 C1 Mix 10 m NA Animal shelter A/canine/NewYork/147926-5/2006 18-Dec-06 C2 Rottweiler 3 yr NA Animal shelter A/canine/Pennsylvania/10909/2007 30-Jan-07 E2 Mix 6 m M Pet A/canine/Pennsylvania/10915/2007 30-Jan-07 E1 NA 6 m F Pet A/canine/Pennsylvania/16699/2007 15-Feb-07 C1 German shepherd Humane Society A/canine/Pennsylvania/94930-3/2007 21-Jul-07 E2 Labrador 3 yr F Pet A/canine/NewYork/115719/2007 7-Sep-07 E1 NA NA MN Pet A/canine/Colorado/6723-8/2008 17-Jan-08 E1; E2 NA NA NA Kenneled dog from Lee County A/canine/NewYork/51854/2008 24-Apr-08 E1 Pitbull 2 yr M Animal shelter A/canine/Pennsylvania/137154/2008 28-Oct-08 C3 Mix 2 yr M Pet A/canine/NewYork/145353/2008 18-Nov-08 C1 NA NA NA Animal shelter A/canine/NewYork/158402-1/2008 31-Dec-08 C2 NA 6 yr M Animal shelter A/equine/Texas/117793/2005 6-Oct-05 C3; E2 Quarter horse 1 yr NA Pet A/equine/Virginia/131054-3/2005 8-Nov-05 E2 NA 3 yr F Equine clinic A/equine/Ohio/113461-1/2005 2005 E2 Quarter horse 2 yr M University equine stable A/equine/Ohio/113461-2/2005 2005 C2; E2 Quarter horse 2 yr M University equine stable A/equine/Ohio/113461-3/2005 2005 E2 Quarter horse 2 yr M University equine stable A/equine/NewYork/146066/2007 26-Sep-07 E2 NA NA NA Equine farm A/equine/Montana/9233/2007 16-Nov-07 C3 Quarter horse 18 yr F Equine training school

a NA: not available; m: month; yr: year; M: male; F: female; N: neutered; S: spayed. b Fatal case. P. Rivailler et al. / Virology 408 (2010) 71–79 73

Fig. 1. Evolution of the CIV genome: phylogenetic analysis of H3 hemagglutinin (HA) and N8 neuraminidase (NA) genes. Phylogenetic analysis of CIV and EIV HA (panel A) and NA (panel B) genes representative of a total of 36 viral genomes sequenced in this project (accession numbers shown in Supplementary Table 1). The phylogenetic tree was constructed by Neighbor-Joining using MEGA software (Kumar et al., 2004) and includes selected EIV H3N8 collected after 1989 as well as CIV sequences. The trees were rooted to A/equine/ Miami/1/1963. The reliability of the phylogeny was assessed with 1000 bootstrap replicates. Values greater than 90% are indicated at the node of the branches. Sequences from this project are indicated with an asterisk (*). The horizontal bar denotes the unit scale of nucleotide substitutions per site for the tree branch lengths. Genes from emerging CIV isolated in 2003–2004 are shown in blue shading, whereas those of enzootic CIV isolated after 2005 are indicated in red shading. EIV isolated in 2005–2207 are indicated in yellow shading. EIV genes most closely related to those of emerging CIVs are indicated with a red arrowhead. equine tree trunk (Figs. 1 and 2, blue shading). Their minimal bootstrap support than the contemporary canine virus clades but are divergence from the equine genes also suggested that they may be the definitely distinct from earlier viruses. In contrast, the genes of some earliest known H3N8 viruses adapted to horizontal transmission in recent equine viruses from Asia (e.g. A/equine/Hubei/6/2008; A/ dogs; thus, referred to as “emerging CIV” in this communication equine/Xinjiang/1/2007, Fig. 1) formed a distinctive clade branching (Crawford et al., 2005; Payungporn et al., 2008). The EIV isolates most away from viruses from North America, Asia, Africa and Oceania closely related to these emerging CIV can be readily identified in each (Figs. 1 and 2). of these trees and may represent the nearest equine viruses sharing an A comparative analysis of tree topologies from each of the CIV and immediate ancestor with them, namely, A/equine/Kentucky/9/2004 EIV genes in the context of a broad range of previously reported (for HA), A/equine/Ohio/1/03 (for NA, PB2), A/equine/Wisconsin/ influenza viruses adapted to other species indicated that all CIV and 1/03 (for PB1, NP), A/equine/California/8560/02 (for PA and NS), A/ EIV genes have co-evolved independently; i.e. no evidence of genetic equine/California/4537/97 (for M) (Figs. 1 and 2; red arrowhead). reassortment was found (Figs. 1 and 2 and Supplementary Fig. 1). One or more EIV with a similar genotype circulating in 2002–2003 Further studies will be necessary to identify the underlying ecological would be a likely candidate ancestor of the emerging A/canine/ or virological determinants of their evolution. Florida/242/03 and/or A/canine/Florida/43/04 viruses. The consistent monophyletic evolution of the CIV isolated from Phylogenetic analysis of seven EIV isolated between 2005 and dogs between 2005 and 2008 (Figs. 1 and 2, red) contrasts with the 2008 from four major geographic regions of the United States graded evolution of the emerging canine viruses (Figs. 1 and 2, blue) indicated that all genes diverged from the equine viruses of previous as indicated by their proximity to contemporary equine viruses. This years (Figs. 1 and 2, yellow shading). These clades have lower evolutionary distance could also be attributed to temporal 74 P. Rivailler et al. / Virology 408 (2010) 71–79

Fig. 2. Evolution of the CIV genome; phylogenetic analysis of PB2, PB1, PA, NP, M, and NS genes. Phylogenetic analysis of CIV and EIV PB2, PB1, PA, NP, M and NS genes (Panels A through F, respectively) representative of a total of 36 viral genomes sequenced in this project (accession numbers shown in Supplementary Table 1). Annotation conventions are as in Fig. 1. Bootstrap values less than 90% are indicated in parenthesis at the node of equine and canine branches. ca: canine; eq: equine. relationships allowing additional generations for recent CIV as well as canine influenza virus clade are not a leading cause of respiratory insufficient sampling. In addition, the ecological separation between disease in US horses (Figs. 1 and 2). Taken together, the eight gene the racing greyhound populations that apparently supported the phylogenies showed that the divergent evolution of canine and emerging CIV and the subsequent circulation of CIV in boarding equine influenza virus genes since 2004 is consistent with separate kennels and pet dog shelters could be a contributing factor. The chains of virus transmission responsible for acute respiratory disease continued circulation of CIV in certain canine populations since 2005 in the respective equine and canine host populations. indicates that the virus has become enzootic in dogs at these locations. Therefore, we refer to the 2005–2008 CIV clade as “enzootic CIV” to Evolution of the CIV and EIV proteomes distinguish it from the “emerging CIV” viruses from 2003 to 2004 (Figs. 1 and 2, red shading). Previous studies identified a set of 6 amino acid substitutions in None of the genes of viruses isolated recently from horses had the the HA protein of emerging canine influenza viruses, thought to be genomic sequence signatures from the 2005 to 2008 CIV clade associated with the host switch event (Crawford et al., 2005; (Payungporn et al., 2008), suggesting that H3N8 viruses from the Payungporn et al., 2008). To characterize the subsequent evolution P. Rivailler et al. / Virology 408 (2010) 71–79 75

Table 2 Evolution of the CIV proteome.

a For HA, amino acid numbering is that of the human H3 hemagglutinin; e.g. A/Aichi/2/68, GenBank accession no. AAA43178. b Amino acid residues found in all H3N8 EIVs collected between 1990 and 2003. Variations to consensus are indicated in footnotes. c One isolate has an asparagine at position 27 (A/equine/California/4537/1997). d Seven isolates (2896 analyzed sequences; 0.2%) have a serine at position 83 (A/equine/Newmarket/1/1993; A/equine/Grobois/1/98; A/equine/Brescia/1999; A/equine/ Cheltenham/1/01; A/equine/Lincolnshire/1/2002; A/equine/Pulawy/1/2005; A/equine/Aboyne/1/05). e One isolate has a serine at position 118 (A/equine/Newmarket/1/1993). f Four isolates (2893 analyzed sequences; 0.1%) have a leucine at position 222 (A/Equine/Alaska/1/91; A/equine/Alaska/29759/1991; A/equine/Rome/5/91; A/equine/ Qinghai/1/1994). g Forty-two isolates (1.4%) have an arginine and 3 isolates (0.1%) have a glycine at position 261. h One isolate has a lysine at position 483 (A/equine/Guelph/G03-0250/2003). i Two isolates (4%; [A/Equine/Alaska/1/91; A/equine/Alaska/29759/1991]) have a methionine at position 147. j Amino acid residues in emerging CIVs (A/canine/Florida/242/2003 and A/canine Florida/43/2004). k Blue-shaded boxes denote novel amino acid substitution in “emerging H3N8 CIV” viruses. l Amino acid residues found in enzootic H3N8 CIV sequences (absent in Florida isolates from 2003 to 2004). m Green-shaded boxes denote novel amino acid substitution in enzootic H3N8 CIV. n One isolate has a valine at position 374. o One isolate has a mixed base isoleucine/methionine at position 29. of canine influenza virus proteomes relative to EIV sequences, we in virus-host interactions favoring viral adaptation to the new canine compared the amino acid sequences of the 11 viral proteins host merit further study. (Supplementary Table 2) from all the new viruses identified in the The proteomes of EIV isolated between 2005 and 2007 revealed course of this study. The multiple amino acid sequence alignment considerable evolution relative to their immediate ancestors. A total of confirmed the 6 previously reported amino acids in HA that 10 substitutions were identified in the proteomes of 2005–2007 EIVs characterized the emerging CIV as distinct from the EIV ancestors compared to those of progenitor viruses circulating up to 2003 in the (Crawford et al., 2005; Payungporn et al., 2008). The whole USA. With the exception of HA, NP, M and NS2, all viral proteins proteome analysis identified 4 additional changes in emerging CIV: acquired amino acid substitutions. The polymerase genes featured a 1 in PB2 (L374I), 2 in PA (D27N, N675D) and 1 in NP (D375N). The large number of substitutions, as follows PB2 I398V, I411V, and latter substitution is found only in one of the two emerging CIV K660R; PB1 F94L, and K621R; PB1-F2 S63Y, and PA I465V. The NA isolates (Table 2 and Supplementary Table 3). Interestingly, none of protein acquired two substitutions; I8M and V35A. The M2 protein the 10 changes identified in the proteomes of emerging CIV reverted showed a single amino acid change; L59M. Interestingly, the 10 amino to the EIV-like amino acid residues in subsequent years (Table 2, acid changes that characterized the 2005–2007 EIV are not found in Supplementary Table 2). All the enzootic CIVs share a total of 9 new emerging and enzootic CIV. Notably, a stop codon introduced at amino acid substitutions as compared to the emerging CIVs (Table 2 position 220 in NS1 from EIV of the so-called “American lineage” was and Supplementary Table 3). These substitutions were not evenly stably maintained in all the EIV analyzed in this study (Table 3). distributed throughout the proteome; changes were more frequent Although these findings are consistent with the continued divergent in PB2 and HA than in other proteins. Three shared substitutions evolution of the EIV clade in the USA, the absence of “fixed” occurred in PB2; S107N, A221V and I292T, and 3 in HA; L118V, substitutions in HA was unexpected. K261N, and G479E (using the human H3 HA amino acid position numbering system, per Table 2)(Payungporn et al., 2008). The 3 Discussion remaining substitutions were observed in NA (I62L, V147I) and PB1 (V200I). In addition, two sites with polymorphic residues selected The presence of canine influenza virus (CIV) in the companion for a single residue were identified: I/M29M in HA and D/N375N in animal population was not identified until the summer of 2005 with NA (Table 2). The amino acid substitutions in virion surface proteins the isolation of CIV from several pet dogs in Florida and the and two polymerase complex subunits are likely to carry functional identification of CIV in the New York City area (Dubovi and Njaa, significance as they are most frequently associated with host range 2008). The movement of CIV in dogs was tracked by the isolation of and virulence (Neumann and Kawaoka, 2006). Their potential roles virus from clinical cases and by the detection of antibodies to CIV in

Table 3 Evolution of the EIV proteome.

Protein PB2 PB1 PB1-F2 PA N8 M2 NS1

Position 398 411 660 94 621 63 465 8 35 59 220 EIV (1990–2004) I I K F K S I I V L R/STOP a EIV (USA, 2005–2007) V V b R c L R Y V M A M STOP CIV I I K F K S I I V L R

a The stop codon first detected in 2002 in the NS1 coding region results deletion of the PDZ domain. b One isolate has isoleucine at position 411 (A/Equine/NewYork/146066/07). c One isolate has a mixed base encoding lysine/arginine at position 660 (A/Equine/Ohio/113461-2/05). 76 P. Rivailler et al. / Virology 408 (2010) 71–79 convalescent animals. When antibody positive dogs were detected, Interspecies transmission of EIV into dogs in 2003–2004 did not attempts were made to obtain clinical samples from contact animals depend on substitution E627K, and this residue remains unchanged for isolation of the virus. The movement of CIV in the companion after 5 year of CIV circulation in the canine population. Lack of animal population was unpredictable and sporadic. By the end of positive selection for PB2 627 K in equine and canine species 2006, there were at least three enzootic areas in the US—Florida, suggests that host factors from these two species interact most Colorado, and the New York City area which included northern New favorably with glutamic acid at residue 627 to support the efficient Jersey and Connecticut. Movement of infected dogs from these areas replication of these virus groups. However, it is likely that preference initiated local outbreaks of infection in California, Wyoming, Ken- for E or K residues at position 627 is context-dependent; i.e. other tucky, Delaware and western Pennsylvania. In each instance, there polymerase complex gene lineages may have other structure- was no evidence that the virus was maintained in the new locations. function properties (Bussey et al., 2010; Coloma et al., 2009; Hatta By the end of 2008, the enzootic area on the East coast had extended et al., 2001; Kuzuhara et al., 2009; Massin et al., 2001; Naffakh et al., into eastern Pennsylvania. The geographic separation of the enzootic 2000; Shinya et al., 2004). areas do present the possibility for CIV to evolve somewhat Previous sequence analysis of the HA from emerging CIV identified independently in each of these regions. Future surveillance in dogs five amino acid substitutions at invariant or highly conserved might reveal spatially defined CIV lineages. positions of the EIV HA; N54K, N83S, W222L, I328T, N483T (Crawford Both cell culture and egg inoculation were successful in isolating d et al., 2005; Payungporn et al., 2008). The substitution of tryptophan CIV and EIV, and in parallel testing neither test system proved at position 222 (W222) of the HA with leucine (W222L) provides the superior to the other (data not shown). The most critical factor in most compelling association with host switch events. All sequenced isolating influenza viruses was early collection of clinical samples after HA from EIV reveals tryptophan at position 222, except A/equine/ onset of clinical signs. In experimental infections, the peak of virus Qinghai/1/1994 and A/equine/Alaska/29759/1991, in which it was shedding is 2–4 days post infection (Deshpande et al., 2009). By day 7 replaced with leucine as well. Interestingly, the W222L substitution post infection an HI antibody response is detectable and infectious present in all the CIV was also temporally correlated with the virus is absent from the respiratory tract. A positive PCR signal can transmission of avian influenza viruses H3N2 to dogs in Korea (Song be obtained for several days longer, but by 7 days after the onset of et al., 2008) as well as the recent transmission of EIV H3N8 to swine in clinical signs, all virus culture tests will be of questionable value. China (Tu et al., 2009). The W222L substitution may alter the receptor The frequent isolation of CIV from dogs with respiratory disease binding functions that mediate favorable interactions with host between 2005 and 2008, suggested its sustained circulation in the factors for virus transmission between dogs. For example, Meisner United States for at least 5 years since its emergence. However, a et al. (2008) reported that an H3N2 human virus with a Y98F complete molecular analysis of viral isolates was necessary to substitution in the receptor binding site is highly attenuated for mice determine whether these influenza isolates originated from dogs but a compensatory W222R substitution restored virulence and infected with emerging CIV, or multiple independent events of EIV receptor binding functions. Finally, a recent report suggested transmission to dogs, which has been detected in nature and inefficient binding of CIV to N-glycolyl neuraminic acid glycans as experimentally (Daly et al., 2008; Kirkland et al., 2010; Yamanaka compared to EIV, suggesting a possible involvement of W222L in host et al., 2009). Our results show that CIV isolated from dogs with acute range functions (Yamanaka et al., 2010). The I328T substitution is respiratory disease between 2005 and 2008 have evolved as a distinct adjacent to the R at the cleavage of the HA precursor molecule HA0 monophyletic lineage, rooted in the emerging CIV from 2003 to 2004 (sequence: PEKQTR↓GIFGAI; T underlined, arrow at cleavage site), and closely related EIV. The absence of equine H3N8 viruses in the CIV which is required to activate virus infectivity (Klenk et al., 1975). lineage suggests that transmission of EIV to dogs was not a major Replacement of a hydrophobic isoleucine with a polar threonine in the cause of acute respiratory disease in the canine populations included HAs of CIV may optimize extracellular cleavage of HA0 in the canine in this surveillance. Conversely, despite the limited number of EIV airway. Interestingly, the EIV transmitted to swine in China (e.g. A/ analyzed, H3N8 viruses isolated from horses are exclusively from the swine/Anhui/01/2006), also acquired this substitution relative to recent EIV clade, suggesting that CIV are not being transmitted back to contemporary equine viruses (Tu et al., 2009). The functional horses. This finding is consistent with the inefficient replication of CIV significance of other substitutions at positions 54, 83, and 483 in the in horses reported recently (Yamanaka et al., 2010). Taken together, mature HA also highly conserved in equine viruses remains unknown. these findings indicate that barring the existence of an unknown The enzootic CIV HA clade also acquired L118V and K261N animal reservoir, CIV has been maintained by horizontal transmission substitutions as compared to the emerging CIV. These changes are in the canine population of the United States for at least 5 years. located near or within known antigenic sites of the human H3 HA The divergence of CIV genes revealed by nucleotide sequence trimer, and may represent early antigenic drift events. Compared to phylogenies entailed multiple non-synonymous mutations that human influenza viruses (H3N2), the CIV HA evolved at a consider- characterized the evolution of the viral proteomes. Nineteen amino ably lower rate (~1.8 aa per year) since its emergence in dogs (Bean acid codons that were highly conserved in the previous decade of et al., 1992). However, it evolved at a faster rate than the EIV EIV H3N8 evolution were mutated in the enzootic CIV proteins counterpart (Lai et al., 2001), as it might be expected for a virus (Table 2). The PB2 and HA proteins showed the greatest number of following a host switch event (Parrish and Kawaoka, 2005). Therefore, changes. Evolution of the contemporary CIV PB2 included multiple it is anticipated that the newly developed canine influenza vaccines substitutions relative to the EIV ancestors; S107N, A221V and I374V. will not require the frequent antigen composition update typical of The former two changes map to PB2 domains of unknown function, human influenza vaccines (Wood, 2002). whereas the last are located in regions mediating cap binding or Although the laboratory hosts used for the isolation of influenza close to the mRNA cap binding site (Guilligay et al., 2008), viruses are known to often mediate selection of variant viruses with suggesting that these substitutions may be involved in optimizing mutations in the HA (Bush et al., 1999; Katz et al., 1987; Robertson essential interactions of the viral polymerase complex with canine et al., 1995; Rogers et al., 1985), there was no correlation between the host factors in the nuclear environment. Previous studies have laboratory host for virus isolation and amino acid substitutions in the shown that glutamic acid to lysine changes at position 627 (E627K) HA from this study, suggesting that this is not a likely source of the of PB2 are important for replication of avian influenza viruses in observed mutations (Table 2 and Supplementary Table 2). mammalian hosts (Hatta et al., 2001). Glutamic acid 627 (627E) has Other changes in the proteome of enzootic CIV were noted in PB1 been highly conserved in the PB2 of equine viruses since their (V200I), and NA I62L. Although these residues were highly conserved introduction from birds into horses in 1963 (Shinya et al., 2007). among EIV sequences (Supplementary Table 2), the role of these P. Rivailler et al. / Virology 408 (2010) 71–79 77 changes, if any, in host adaptation would be restricted by the Diagnostic Center at Cornell initiated an enhanced testing program similarity between these hydrophobic amino acids (Ruigrok et al., on respiratory samples from dogs that were submitted by referring 2010). No novel changes were noted among the proteins encoded by veterinarians. Most of the specimens received were nasal swabs, NP, M and NS genes of enzootic CIV. either cotton or Dacron, or an occasional tissue sample from We also examined the evolution of the proteome of EIV in the necropsy. In addition to routine virus isolation attempts for standard decade before the emergence of CIV for clues regarding changes in respiratory viruses of dogs, samples were also subjected to real-time proteins involved in host interactions. The most remarkable change reverse transcriptase PCR assay for influenza virus targeting the was found in the NS1 protein. EIVs isolated after 2002 contain matrix gene (Spackman et al., 2002). This assay that was being used truncated NS1, with a premature termination codon at position 220 for avian influenza virus surveillance programs detected all canine (Bryant et al., 2009). This deletion resulted in the loss of a PDZ and equine influenza viruses tested. The standard protocol for canine domain, a widely distributed protein motif that mediates protein– and equine samples was to perform the RT-PCR assay and those protein interactions in cell signaling and modulates influenza virus samples with Ct values of b40 were processed for influenza virus replication (Jackson et al., 2008; Jemth and Gianni, 2007). However, isolation. Screening of equine respiratory samples for influenza virus the EIV ancestor of CIV had a full-length NS1, and all CIV NS1 proteins using the same strategy had been ongoing prior to the initiation of retained the PDZ domain. Further studies on the importance of NS1 testing in canines. PDZ domain of EIV for infection and sustained transmission in dogs Influenza viruses were isolated by two methods; cell culture would shed light on this topic (Kirkland et al., 2010; Yamanaka et al., isolation and/or embryonated chicken eggs. For tissue culture 2009; Yamanaka et al., 2008) (Supplementary Table 4). isolation, MDCK cells were inoculated with the test sample Sustained transmission of CIV (H3N8) in dogs was reported only in followed by incubation at 37 °C in medium containing 0.3% BSA the United States (Dubovi and Njaa, 2008; Payungporn et al., 2008) plus 2 μg/ml of acetylated trypsin (Sigma). Culture supernatants whereas EIV (H3N8) outbreaks have been reported globally after its were tested for the presence of influenza virus by either emergence in horses decades ago. The apparent limited global spread hemagglutination (HA) assay, RT-PCR, or antigen-capture ELISA of CIV contrasts with the global distribution of other viral respiratory tests. For egg isolations, 10-day-old specific pathogen-free (SPF) diseases of dogs. It has been suggested that CIV is not as transmissible embryonated eggs were inoculated in the allantoic cavity with 0.1– as other influenza viruses (Dubovi and Njaa, 2008). However, global 0.2 ml of clinical sample. Eggs were incubated for 4 days and the surveillance for CIV infections in dogs is incomplete and further allantoic fluid collected. Presence of influenza virus was deter- studies are needed to better understand the geographic distribution of mined by HA assay. Negative samples were re-inoculated into eggs this virus. In addition, ongoing virologic surveillance programs for EIV for a second passage. HA negative samples from the second should identify cases of respiratory disease in horses caused by CIV, if passage were considered negative for influenza virus. In many they were to occur in significant numbers. instances, if one of the isolation systems yielded a positive result, Natural infection of dogs with the 2009 pandemic H1N1 virus were the other was not attempted. reported by the news media (AVMA, 2009) suggesting that reassortment between the human pandemic virus and H3N8 CIV could Sequence analysis theoretically occur in co-infected dogs. Dogs were also reported to be susceptible to infection with human H3N2 influenza viruses (Rom- RNA isolation and sequencing procedures were described previ- omvary et al., 1975). Although the probability of co-infection between ously (Crawford et al., 2005; Payungporn et al., 2008). Briefly, viral human influenza viruses and CIV seems low due to the restricted RNA was extracted from infectious allantoic fluid or cell culture circulation of the latter to dog shelters, and boarding kennels housing supernatants with the Qiagen RNeasy extraction kit, and a one-step many animals, virologic surveillance and molecular analysis of RT-PCR kit (Qiagen) was used to transcribe and amplify genomic circulating isolates from canine species is essential for early detection segments as DNA fragments. DNA amplicons spanning the complete of emerging reassortment. open reading frame(s) of viral genes were sequenced on an Our passive virologic surveillance based on acute respiratory automated Applied Biosystems 3730 system using cycle sequencing disease indicated that CIV were isolated exclusively from dogs and dye terminator chemistry (Perkin-Elmer, Foster City, CA). Primer equine viruses from horses. Several factors could account for the sequences are available upon request. Contiguous sequences were linkage of these viruses to the homologous species. First, interspecies generated with Sequencher 4.9 software (Gene Codes). The overall transmission does not occur due to the limited contact between the sequence coverage was estimated at 4.3-fold. Gene sequences urban canine population in which CIV circulates and the rural equine generated in this study were submitted to the GenBank database population. Second, interspecies transmission of canine and EIV does (Supplementary Table 1). occur, but fails to cause clinically apparent disease or its incidence is below the detection threshold of this study. Third, interspecies Phylogenetic analysis transmission does not occur due to viral dependence on a mutually exclusive species-specific function. Further studies are needed to Our analysis data set included influenza gene sequences that determine the circulation dynamics of H3N8 influenza viruses in were downloaded from the NCBI Influenza Virus Resource (http:// canine and equine hosts. However, these findings provide a rationale www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Curated data sets to postulate that H3N8 EIV circulating in horses do not easily infect for each gene were aligned to newly generated project sequences canines and spread widely in this species and vice versa. In general, it using MAFFT software (Katoh et al., 2002). Phylogenetic tree would be important to determine whether interspecies transmission inferences were generated by the MEGA software package (version of H3N8 influenza viruses has a fitness cost that prevents these 4) using the neighbor-joining method with a maximum composite viruses from circulating in dogs and horses indiscriminately. likelihood model (Kumar et al., 2004; Saitou and Nei, 1986). 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