TECHNICAL BULLETIN November 2018 Lessons from the Characterization of Canine Immunity to

Karen Stasiak, MSN, DVM Veterinary Medical Lead, Biologicals Zoetis Petcare, Veterinary Specialty Operations Parsippany, NJ Annette Litster, BVSc PhD FANZCVS (Feline Medicine) MMedSci (Clinical Epidemiology) Senior Veterinary Specialist Zoetis Petcare, Veterinary Specialty Operations

Key Points: • There are two known circulating strains of CIV in the United States, CIV H3N8 and CIV H3N2 • Approximately 80% of dogs exposed to CIV develop clinical signs • Estimated mortality rates vary from 5-10% • Canine influenza has reassorted with human and swine • CIV contains virulence factors that promote neutrophilic infiltrates into the lung and are similar to the virulence factors in known severe avian and human influenzas • CIV is capable of deep penetration into the lower airways where pro-inflammatory cytokines and apoptosis can cause pathology

ntroduction: Canine influenza is and working dogs, and could Ian emerging around the potentially become a zoonotic threat. world that was first identified in 2004. The aim of this paper is to summarize Two known strains of canine influenza the evolution of CIV; describe its virus (CIV) circulate presently and clinical significance; and to evaluate large scale outbreaks continue. Viral its effects on the canine immune reassortants have been identified system, comparing and contrasting over the last decade and canines have this to human influenza infections. shown susceptibility to human, avian By understanding the immune and swine influenzas. Canine influenza response to CIV, we gain an poses health risks to beloved pets appreciation for how disease pathogenesis results in clinical signs and how These factors facilitate the continued spread of the disease, comorbidities could impact disease presentation. especially in pet dogs living highly social lifestyles and dogs kept in multi-dog housing, such as boarding kennels This understanding may assist veterinarians in and shelters. Approximately 80% of dogs exposed to CIV determining the importance of building herd develop clinical signs including fever, nasal and ocular immunity with widespread vaccination and discharges, and a cough that may persist for up to 3 weeks. helping to identify at-risk canine patients. A Estimated mortality rates vary from 5-10%, but have been 9 literature review was conducted covering the as high as 36% in some CIV H3N8 outbreaks. CIV H3N8 has not been shown to transmit to other species. Experimental years 2005-2018 with search terms including challenge of horses with CIV H3N8 shows seroconversion ‘canine influenza’ and ‘immune response’. but lack of disease or viral shedding.10 CIV H3N2 has been shown to be contagious to cats, with one Korean Evolution of CIV shelter reporting 100% morbidity and 40% mortality.11-12 There are two known circulating strains of CIV in the United Experimental challenge confirms that cats are susceptible States, CIV H3N8 and CIV H3N2. These viruses are capable to CIV H3N2 and can transmit infection to other cats, of dog to dog transmission which distinguishes them as resulting in clinical disease.13 canine. CIV H3N8-induced disease was first identified in racing greyhounds in 2004 and is of equine origin1, Influenza A Susceptibility stemming from a mutation at the hemagglutinin receptor and Reassortants biding site which allowed the virus to adapt its host species Influenza A viruses are single stranded RNA viruses 2 from horses to dogs. CIV H3N8 spread rapidly throughout in the family with 8 gene segments the racing greyhound community, typically causing fever, encoding for structural and nonstructural proteins. cough, and nasal discharge in infected dogs; however (Figure 1) These viruses are divided into subtypes based some dogs died peracutely due to pulmonary hemorrhage on the hemagglutinin (H) protein, which is responsible for associated with suppurative bronchopneumonia. CIV H3N8 attachment and infection of cells; and the neuraminidase was first identified in a pet dog in 2005 and (N) protein which is responsible for cleavage of sialic acid serologic evidence showed a spread to 25 states over 3 and release of viral particles from infected cells. There are 1 years. Currently, CIV H3N8 seropositivity rates vary widely 18 known H types and 11 known N types. The ability of the 3-4 by geography in 39 states and range from 2.3-15%. hemagglutinin to bind is host-specific. However, given the Of avian origin, CIV H3N2 was first identified in pet dogs ability of RNA replication to develop mutations, antigenic in Chicago in 2015, although it has been circulating drift can occur, allowing infectivity into new host ranges. in SE Asia since 2005. Sequence analysis of the virus In addition, co-infection with more than one influenza demonstrated that the Chicago strain was most similar A virus may allow for gene , resulting in an 5 to CIV H3N2 circulating in South Korea during 2015. antigenic shift and the ability to infect new host species. Widespread outbreaks continue across the United States. Host viral receptors vary by species and play a role in Further sequence analysis has linked some U.S. CIV H3N2 species-specific susceptibility to influenza. Tracheal viral outbreaks to ongoing importation of infected dogs from receptors in birds, horses, and dogs consist of α2, 3 sialic 6 Korea and China. acid whereas human tracheal viral receptors consist of α2, 6 sialic acid. Swine express both receptors and serve Clinical Significance of CIV as the “mixing vessel” when co-infected with avian and Transmission dynamics of CIV H3N2 differ from CIV H3N8. human influenzas.14-16 Although dogs predominantly express CIV H3N8 has a short viral shed window from 1-7 days, with α2, 3 sialic acid receptors, some foci of α2,6 sialic acid peak viral shedding occurring between days 2-47, whereas receptors have been identified in the respiratory tract,17 CIV H3N2 has been shown to shed intermittently at low which may allow the dog, like the pig, to serve as a “mixing levels by some dogs for at least 21 days.8 Viral shedding vessel”. This potential zoonotic risk is one of the greatest kinetics underpin the timing of diagnostic testing and have challenges that has emerged from the establishment of implications for biosecurity. For both CIV H3N8 and CIV CIV in the pet population. It has been demonstrated that H3N2, dogs shed virus prior to the onset of clinical signs dogs may be infected not only with CIV H3N8 and CIV and influenza A viruses survive on surfaces for 24-48 hours. H3N2, but also with pandemic H1N1, highly pathogenic

2 (HPAI) H5N1, avian influenza H9N2,human chemokine response to CIV. In general, human and swine H3N2, as well as reassortant H3N1 and H5N2.14, 18 CIV H3N2 chemokine responses to influenza show elevations in IL-6, reassorted with pandemic H1N1 to create H3N1 in South which is responsible for the development of certain acute Korea. This virus is capable of causing nasal shedding phase proteins.25 and mild pulmonary histopathologic changes in dogs.19 A CIV H3N2 infection also generates elevated levels of CIV H3N2 isolate recovered from a naturally infected dog interferon (IFN)-γ and tumor necrosis factor (TNF)-α.23 revealed reassortment with pandemic H1N1 and expressed IFN-γ and TNF-α have antiviral activities and have been the Matrix gene (M) segment from H1N1. Challenge identified as the pro-inflammatory cytokines in human studies in dogs using this virus demonstrated its ability influenza infection.26 TNF-α demonstrates species- and to cause clinical disease by direct and indirect exposures virus-specificity, but may not be released in all influenza and also to induce seroconversion and viral shedding. infections.27 There is evidence that CIV generates Similar recombinations with and pandemic 20 macrophage activation through the classical IFN-mediated H1N1 have also been reported. Additional samples from pathway resulting in TNF-α production, but innate activation CIV H3N2-infected dogs showed 23 different genotypic of macrophages also occurs, as evidenced by increases in patterns of recombination with CIV H3N2 and pandemic macrophage receptors with collagenous structure (MARCO) H1N1, with canine H and human M proteins most commonly during infection.28 CIV H3N8 also has been shown to expressed. Some of these genotypes caused severe clinical 21 replicate in alveolar macrophages and stimulate high levels disease in experimentally infected mice. Surveillance of of TNF-α.29 These immune responses are important influenza viruses in China over a 2-year period identified in limiting viral infection. swine, human and canine influenza reassortants, with two genotypes capable of further infecting swine, humans, and In a mouse model of human influenza, alternatively 22 dogs. The ability of CIV to reassort with other influenza A activated macrophages (AAM) contributed to increasing the viruses, the demonstrated susceptibility of dogs to a variety susceptibility to bacterial during the recovery of influenza viruses, and the close relationship between pet phase after influenza infection. The late appearance of these dogs and their owners, poses risks for the development macrophages represents a change in the cytokine milieu of zoonotic influenza. from inflammatory to maintenance. The transition to a T helper 2 (Th2) response and the secretion of IL-4 and IL-13 The Innate Immune Response to CIV decreases the bactericidal activity of macrophages. AAMs The difference between dogs that develop mild clinical secrete arginase-1, which competes with inducible nitric disease and those that progress to severe disease or oxide synthase, which known for its antibacterial effects.30 mortality has often been attributed to co-infection with Although the role of AAMs is unknown in CIV, it is possible secondary bacterial pathogens. A deeper understanding of that altered cytokine expression increases the likelihood the immune response and the impact of comorbidities may of secondary bacterial infection, which could contribute shed light on additional factors that contribute to disease to severity of disease. The presence of parasitic infection severity, mortality, and chronic sequela. A key difference in or pulmonary migrating larval stages are common in dogs the canine response to influenza compared to that and may set the stage for a predominant Th2 response. in humans is the secretion of interleukin (IL)-8. CIV H3N2 This could then lead to the development of a suboptimal infection stimulates marked increases in IL-8 in the serum antiviral immune response, thereby complicating the course on days 3 and 6 after intranasal experimental challenge. of disease. The role of chemokine IL-8 is to recruit neutrophils.23 CIV H3N2 has been shown to induce neutrophilic infiltration into It has been shown that CIV H3N2 infects tissues from the lungs without secondary bacterial co-infection.24 There the upper and lower respiratory tract, the bronchiolar 23 are also elevated levels of monocyte chemotactic protein epithelium and type I pneumocytes. The ability of CIV (MCP-1) in the lungs post-infection. The role of chemokine H3N2 to penetrate the lower respiratory tract and cause MCP-1 is to recruit monocytes. (Figure 2) Histopathology of tissue destruction likely contributes to the long-term CIV H3N2 infected lungs shows mild to severe pneumonia sequelae of CIV infection. Apoptosis may also be a factor with neutrophilic and lymphocytic infiltrates, pulmonary in tissue damage related to CIV infection. MicroRNAs are hemorrhage, and neutrophils and macrophages in the small molecules that regulate many cellular functions and alveoli.23 These results are consistent with the known pathways, including response to viral infection. Differential

3 upregulation and downregulation of microRNAs have through the NF-κB pathway. PB1-F2 has also been been demonstrated in dogs experimentally infected implicated in apoptosis. PB1-F2 inflammatory residues have with CIV H3N2 and H5N1.31 Additional evaluation of dogs been identified and contribute to lung inflammation and experimentally infected with CIV H3N2 showed an increase pulmonary neutrophil infiltrates. A combination of delaying in microRNA in lung tissue early in infection, and specifically the initial immune response through decreased IFN-β, and identified cfa-miR-143. MicroRNA 143 is known for its role in recruitment of neutrophils and inflammatory cytokines, apoptosis in cancer and the apoptotic pathways upregulated leads to severe clinical disease. This has occurred in avian by cfa-MiR-143 act via p53.32 H5N1 and pandemic human influenzas in 1918, 1957 and 1968. The sequence length of PB1-F2 varies and contributes to its The Adaptive Immune Response to CIV virulence, with the full length variant causing more severe Antibodies generated after infection with influenza virus are clinical disease and increasing susceptibility to secondary critical to the protective response. While secretory IgA is infection. Evaluation of CIV H3N8 isolates identified 65% important for immune exclusion at the mucosal surface, IgM with a full length PB1-F2 accessory protein and 22% with and IgG are capable of pathogen elimination. Additionally, inflammatory residues. CIV H3N2 isolates contained 95% antibody-dependent cell mediated cytotoxicity (ADCC) full length variants and 100% inflammatory residues. By can be induced by influenza A infections.26 Upregulation contrast, human seasonal influenza had <1% with full length 34 of gene expression for IgG Fc fragments in natural killer PB1-F2 proteins and no inflammatory residues. Amino acid (NK) cells have been identified in dogs infected with CIV substitutions in the PB2 gene, most notable E627K and H3N2, suggesting that ADCC plays a role in the immune D701N, have been shown to alter viral virulence in some response to CIV.28 T cell differentiation to CD8+ cytotoxic influenza A viruses. Insertion of these substitutions into 35 T cells (CTLs) in response to virally infected cells is an CIV H3N2 did not affect pathogenicity. Two variants of essential immune response. CD4+ T cell differentiation to nonstructural protein PA-X have been shown to modulate T helper 1 cells drives this response through the secretion the innate immune system. CIV H3N8 and CIV H3N2 of IFN, IL-2 and IL-12 and differentiation to T helper 2 cells have been shown to contain the shorter length PA-X, as 36 promotes B cell-mediated antibody production.26 Controlled does pandemic H1N1. CIV contains virulence factors flow cytometry studies of T cell subsets from the peripheral that promote neutrophilic infiltrates into the lung and blood of CIV H3N2-infected dogs failed to demonstrate are similar to the virulence factors in known severe avian differences between infected and uninfected dogs.23 and human influenzas. However, analysis of lung tissue from CIV H3N2-infected dogs showed gene upregulation in T helper 1 and T helper Summary 2 cells.28 This suggests a balance between cell mediated CIV immune responses promote pulmonary neutrophilic and humoral responses in the immune response to CIV. infiltration. The virus is capable of deep penetration into the lower airways where pro-inflammatory cytokines Viral Interference with Immunity and apoptosis can cause pathology. Additionally, viral Viral infections stimulate a cascade of responses from both interference with innate immunity contributes to successful the innate and adaptive immune system. The recognition of infection and CIV expresses virulence factors that may viral RNA by pathogen recognition receptors, specifically contribute to increased morbidity and mortality. Dogs are RIG-I, results in downstream activation of transcription commonly exposed to multiple pathogens, co-morbidities factors NF-κB and interferon response factors (IRF), that such as parasitic infection, and concurrent therapies such ultimately produce type I IFN, activate the adaptive immune as steroid use, which could negatively impact the immune system, and produce cytokines. However, pathogens come response to infection. Multiple reassortants of CIV, canine armed with virulence factors to circumvent the host immune receptor capability to serve as a “mixing vessel”, and the system. In human influenza, the NS1 viral protein blocks susceptibility of the dog to multiple influenza A viruses several steps in the immune response.26 CIV H3N2 has been could potentially lead to the development of canine-human shown to block IFN-β production by blocking NF-κB and zoonotic influenza. Vaccination against CIV can decrease IRF3.33 (Figure 3) Influenza A accessory protein PB1-F2 viral circulation through canine populations, and reduce has been shown to decrease IFN production by blocking the risk of clinical disease. RIG-I, but increase pro-inflammatory cytokine production

4 Reflection the risk of severe disease in dogs. These factors, combined with the large number of influenza reassortants identified This review describes how the immune responses to CIV over the last decade, generates a sense of urgency can be directly linked to clinical pathology. The canine to prevent the emergence of canine to human zoonotic chemokine immune response to influenza virus is different influenza. Widespread vaccination against CIV to than human responses and explains the severity of clinical prevent transmission and clinical disease is an important signs that can occur, even in the absence of secondary place to start. bacterial infection. The presence of CIV virulence factors that are identical to those in the most severe influenzas increases

NA (Neuraminidase)

Lipid Layer

HA (Hemagglutinin)

M2 (Ion Channel)

M1 (Matrix Protein)

PB1, PB2, PA (RNA Polymerase)

NP (Nucleocapsid Protein)

NEP (Nuclear Export Protein)

Segmented (-) Strand RNA Gene

Figure 1. structure. Enveloped negative sense, single-stranded RNA virus containing 8 gene segments encoding for structural and non-structural proteins.

5 Pro-inflammatory Cytokines IFN-γ Viral Infected Cells CD8+ Cytotoxic MHC I Naïve MHC I T Cell T Cell Type I IFN IL-12

MCP-1 CD4+ IL-1 IL-2 Neutrophils MHC II IFN-γ Dendritic Cell TH1 TNF Memory T Cells MCP-1 TNF-α IL-8 Monocyte Macrophage

CD4+

TH2 IL-4 MHC II IL-13

Naïve BCR Memory Plasma B Cell B Cell Cell IgG

Dendritic Cell IgA

ADCC NK Cell

Figure 2. Innate and adaptive immune responses to CIV infection. Viral antigen presentation through endogenous pathway MHC I stimulates CD8+ cytotoxic T cells (CTL). Antigen presentation through exogenous pathway MHC II stimulates CD4+ T cell differentiation to T helper 1 (Th1) and T helper 2 (Th2) cells. Th1 cytokine expression enhances CD8+ activity. Th2 cytokine expression enhances B cell antibody production. Natural killer cell antibody dependent cell mediated cytotoxicity (ADCC) assists in viral clearance. CIV induces chemokine IL-8 and MCP-1 recruiting neutrophils and monocytes to the lung.

Figure 3. Recognition of viral RNA by cytosolic pathogen recognition receptor retinoic acid-inducible gene I (RIG-I), Viral RNA induces conformational change exposing a critical domain and with mitochondrial antiviral signaling proteins (MAVS) activates downstream signaling, ultimately activating transcription factors NF-kB/IRF inducing IFN genes resulting in Type I IFN RIG-I production and inducing genes for pro-inflammatory cytokines. Canine influenza virus nonstructural protein NS1 blocks activation of NF-kB/IRF. CIV NS1 Blocks NF-κB/IRF

NF-κB/IRF MAVS

IFN Genes

Type I IFN

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