H3N2 canine influenza virus causes severe morbidity in dogs with induction of genes related to inflammation and apoptosis
- Young Myong Kang†1, 2,
- Heui Man Kim†1, 2,
- Keun Bon Ku1, 2,
- Eun Hye Park1, 2,
- Jung Yum1, 2 and
- Sang Heui Seo1, 2Email author
© Kang et al.; licensee BioMed Central Ltd. 2013
Received: 25 February 2013
Accepted: 25 September 2013
Published: 3 October 2013
Dogs are companion animals that live in close proximity with humans. Canine H3N2 influenza virus has been isolated from pet dogs that showed severe respiratory signs and other clinical symptoms such as fever, reduced body weight, and interstitial pneumonia. The canine H3N2 influenza virus can be highly transmissible among dogs via aerosols. When we analyzed global gene expression in the lungs of infected dogs, the genes associated with the immune response and cell death were greatly elevated. Taken together, our results suggest that canine H3N2 influenza virus can be easily transmitted among dogs, and that severe pneumonia in the infected dogs may be partially due to the elevated expression of genes related to inflammation and apoptosis.
The influenza virus infecting vertebrates is categorized as genera A, B, and C on the basis of the antigenic differences in their nucleoprotein (NP) and matrix proteins (M) [1, 2]. Influenza A virus is further categorized according to the 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase (NA) [2, 3]. All the known influenza A viruses can be found in aquatic birds, and have a broad spectrum of hosts, including chickens, cats, dogs, pigs, whales, and humans . The transmission of influenza from animals to humans does not give rise systemically to a pandemic. It is predicted that pandemics caused by influenza A virus may occur 3–4 times per century. During the 20th century, humans experienced three pandemics: an H1N1 pandemic in 1918, an H2N2 pandemic in 1957, and an H3N2 pandemic in 1968 [4–9]. The first pandemic of the 21st century occurred in 2009, caused by the swine-originated H1N1 influenza virus .
Dogs are one of the most popular companion animals and live in close contact with humans. Dogs infected with influenza A viruses, including H3N8, H3N2, and H5N1, have been previously reported [11–13]. An H3N8 influenza A virus was isolated in January 2004 from the lung tissues of racing greyhound dogs that suffered from severe clinical signs such as high fever, hemorrhagic tracheitis, bronchopneumonia, and vasculitis [11, 14]. Further cases of H3N8 infections in racing dogs were reported in 6 US states in the summer of 2004, and in 11 US states in 2005 and 2006 [11, 14, 15]. More H3N8 influenza viruses were isolated from archived tissues of greyhound dogs that died in Florida in late 2003, from greyhound dogs in Texas in 2004, and from 2 pet dogs in 2005 . Fatally infected dogs that had fed on the carcasses of chickens infected with highly pathogenic (HP) H5N1 influenza virus were reported in Thailand . Serological study of 629 village dogs for H5 antibodies in central Thailand showed that some of those dogs were positive for H5N1 infection . These studies were conducted to evaluate dogs as potential vectors in the transmission of HP H5N1 influenza virus to humans [17, 18]. The studies showed that dogs inoculated with the HP H5N1 influenza virus did not show severe clinical signs, but suffered only from conjunctivitis and showed transiently elevated body temperature. All inoculated dogs were seroconverted, and transmission of viruses from inoculated dogs to contact dogs was not observed. An outbreak of avian origin H3N2 influenza virus in pet dogs occurred in South Korea in 2007 . When beagles were infected with the canine H3N2 influenza virus, they suffered from high fever, necrotizing tracheobronchitis, and bronchioalveolitis . Avian origin H3N2 influenza virus was also isolated from clinically ill dogs in southern China in 2006 and 2007 .
In this study, we are interested in finding out the pathogenicity of a recently isolated canine H3N2 influenza virus in dogs. Therefore, we examined the clinical signs of dogs infected with a canine H3N2 influenza virus and the morphology of a recently isolated, avian origin, canine H3N2 influenza virus. We used broad-spectrum microarray analysis and real-time PCR to investigate the possible pathogenesis by which the H3N2 influenza virus infected the dogs.
Materials and methods
A canine influenza virus, A/Korea/S1/Canine/2012 (H3N2), was isolated from a dog showing respiratory clinical signs in an animal clinic in Korea in 2012 using a nasal swab in PBS (pH 7.4) to inoculate a 10-day-old hen egg.
Approximately 2-month-old Korean farm dogs (Korean mongrel, n = 3) were serologically tested using a hemagglutination-inhibition (HI) assay with 0.5% turkey red blood cells. They were negative for human influenza viruses (H1N1, H3N2, and Influenza virus B) and canine H3N2 influenza virus. All animal experiments were performed at a biosafety level 3 (BSL-3) facility approved by the Korean government.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory animals of Korean veterinary quarantine and service. The animal experiments were approved by the Animal Experimental Ethics Committee at the Chungnam National University.
Canine H3N2 influenza virus imaging by transmission electron microscopy
The H3N2 canine influenza virus was propagated in a 10-day-old hen at 35 °C for 60 h. The allantoic fluid containing the virus was harvested and concentrated to one-tenth of the original volume using an Amicon concentrator apparatus. The concentrated viruses were purified using a 20–75% sucrose continuous gradient at 26 000 rpm (4 °C) for 2 h. The purified viruses were fixed in a 2.5% paraformaldehyde-glutaraldehyde mixture buffered with 0.1 M phosphate (pH 7.2) for 2 h, postfixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in graded ethanol and propylene oxide, and embedded in Epon-812. The sample was stained with uranyl acetate and lead citrate, and examined under a CM 20 (Philips, Netherlands) electron microscope.
Measurement of clinical signs and viral titers in infected dogs
The dogs (n = 3) were infected intranasally (i.n.) with 106 EID50 of canine H3N2 influenza virus, and the rectal body temperature and body weight were measured for 14 days after infection. Daily swabs were obtained from the trachea and rectum, suspended in PBS (pH 7.4), and used to inoculate a 10-day-old hen egg to determine the log10 egg infectious dose 50/mL (log10 EID50/mL). The EID50/mL was calculated as previously described . Three other Korean mongrels were mock-infected with PBS as controls.
Clinical scores of infected dogs
Clinical signs were observed in the dogs for 14 days after infection. The clinical scores were the mean score of activity and respiratory signs. Ocular and nasal discharges were scored as follows: 0 for no discharge, 1.0 for serous discharge, 2.0 for mild mucus, and 3.0 for copious mucus. Cough was scored as 0 for no cough, 1.0 for mild cough, 2.0 for moderate or persistent cough, and 3.0 for severe cough accompanied by choking or retching sounds. Their appetite was scored as 0 for present, 1.0 for mild anorexia, 2.0 for mild anorexia and adipsia, and 3.0 for persistent anorexia and depression. Their hair was scored as 0 for normal, 1.0 for mildly rough, 2.0 for moderately rough, and 3.0 for severely rough hair.
Aerosol transmission of canine H3N2 influenza virus in dogs
Aerosol transmission of canine H3N2 influenza virus was performed as described in reference . The 3 experimental dogs infected with 106 p.f.u. of canine H3N2 influenza virus were placed in a cage, and 1 day later, 3 naïve dogs in the cage were placed about 90.0 cm apart from the experimental dogs. The nasal and cloacal swabs in PBS (pH 7.4) were acquired 3 and 5 days after the transmission setup. The viral titers in swabs were determined by EID50/mL.
Imaging of gross lung lesions
The experimentally-infected dogs were euthanized at day 5 post infection using intravenous injection of T61 (0.3 mL/kg of body weight; Intervet, USA). Lung tissues (left cranial lobes) were then collected. The lung tissues of the control dogs were also acquired in a similar manner. Images of the ventral and dorsal lesions of lungs were captured.
Histopathological staining and immunohistochemistry of lung tissues
The lung tissues were fixed by immersing in 10% neutral buffered formalin, and were embedded in paraffin. Five-micrometer-thick sections were made from the paraffin-embedded tissues, and were stained with hematoxylin and eosin (H&E) as described previously . The stained tissues were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).
Five-micrometer-thick sections were stained with mouse anti-influenza A virus nucleoprotein antibody (Serotech, United Kingdom) . The tissue sections were deparaffinized and hydrated in distilled water, followed by fixing with 100% chilled acetone for 2 h for permeabilization. The endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 for 15 min at 37 °C before the sections were blocked with 5% bovine serum albumin in PBS (pH 7.4) for 1 h. The blocked tissue sections were labeled with mouse anti-influenza A virus nucleoprotein antibody (1:1000 dilution) by incubating at room temperature for 1 h. The labeled tissue sections were stained with biotin-labeled goat anti-mouse immunoglobulin (Vector, USA), VECTASTAIN ABC-AP (Vector, USA), and Vector red alkaline phosphatase substrate (Vector, USA). The stained tissue sections were counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA, USA), after which the stained sections were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).
Gene expression and transcription analysis using DNA microarray in the dog lung tissues
Sterile mortars and pestles were used to pulverize about 1 g of each lung tissue sample in liquid nitrogen; the samples were crushed until a fine powder remained. The powder from each sample was placed into a cold 2-mL microtube, and 1000 μL of TRIzol (Invitrogen, USA) was added and mixed by inverting the tube to help in cell lysis. The TRIzol solution containing the disrupted tissue was then centrifuged at 12 000 g for 10 min at 4 °C. The colorless supernatant phase was collected into a new 2-mL tube and incubated in ice for 5 min, after which 200 μL of chloroform (Sigma-Aldrich, USA) was added to the samples, vigorously shaken by hand, and incubated again in ice for 2 min. This solution was centrifuged at 12 000 g for 15 min at 4 °C, and the aqueous phase was collected into a new cold tube. Chilled isopropyl alcohol (500 μL; Sigma-Aldrich, USA) was added to the solution for RNA separation, mixed by inverting the tube, and incubated in ice for 10 min. The solution was centrifuged at 12 000 g for 10 min at 4 °C, after which the supernatant was decanted, and the pellet, recovered. Then, the RNA pellet was washed with 1000 μL of 75% chilled ethanol (Sigma-Aldrich, USA), and the tube was inverted to wash the RNA. The solution was centrifuged at 9000 g for 5 min at 4 °C. The supernatant was then decanted, the pellet was recovered, and was air-dried at room temperature for approximately 10–15 min. RNA was dissolved into 50 μL of DEPC-treated water by pipetting the solution a few times and treated with DNase (Qiagen, USA).
The synthesis of target cRNA probes and hybridization were performed on equal amounts of control and test RNA from each dog using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, USA) according to the manufacturer’s instructions. For each sample, 1 g of total RNA and T7 promoter primer mix was incubated at 65 °C for 10 min. A cDNA master mix (5× First strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mix. The samples were incubated at 40 °C for 2 h and then at room temperature; the dsDNA synthesis was terminated by incubating at 65 °C for 15 min. The transcription master mix was prepared according to the manufacturer’s protocol (4× Transcription buffer, 0.1 M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3-CTP). Transcription of the dsDNA was achieved by adding the transcription master mix to the dsDNA reaction samples, and incubating at 40 °C for 2 h. Amplified and labeled cRNA was purified on a cRNA Cleanup Module (Agilent Technology) according to the manufacturer’s protocol. Labeled cRNA target was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). After checking labeling efficiency, fragmentation of cRNA was performed by adding 10× blocking agent and 25× fragmentation buffer, and incubating at 60 °C for 30 min. The fragmented cRNA was resuspended in 2× hybridization buffer and directly pipetted onto an assembled Agilent Canine Oligo Microarray (44 K) . The arrays were hybridized at 65 °C for 17 h using an Agilent Hybridization oven (Agilent Technology, USA). The hybridized microarrays were washed according to the manufacturer’s washing protocol (Agilent Technology, USA).
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA, USA). All data normalization and selection of fold changes in gene expression were carried out using GeneSpringGX 7.3 (Agilent Technology, USA). The averages of normalized ratios were calculated by dividing the average of normalized signal channel intensity by the average of normalized control channel intensity. Functional annotation of genes was performed according to the Gene Ontology database , BioCarta , GenMAP , DAVID bioinformatics resource , and National center for biotechnology information . GEO accession number is GSE44545.
Quantification of dogs’ genes by real-time polymerase chain reaction (PCR)
Total RNA was collected from lung tissues (1 g) of dogs (n = 3 per group) that were infected with canine H3N2 influenza virus using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One milliliter of TRIzol reagent (Invitrogen) was added to tubes containing tissues and incubated at room temperature for 5 min. Chloroform (200 L) was added and the solution was mixed by vortexing for 15 s and centrifuged for 15 min (12 000 rpm, 4 °C). The upper RNA-containing band was collected and mixed with 500 L of isopropanol (Sigma-Aldrich, St. Louis, MO, USA) in a new 1.5 mL tube. Each sample was centrifuged for 10 min (10 000 rpm, 4 °C), and the RNA-containing pellet was washed with 100 μL of 75% ethanol in water by centrifuging for 5 min (10 000 rpm and 4 °C). The washed RNA was resuspended in 50 μL of diethyl pyrocarbonate (DEPC)-treated water.
Real-time PCR primer sequences
TCA ACG GAT TTG GCC GTA TTG G
TGA AGG GGT CAT TGA TGG CG
TGT TGT TGG GCA ACT GAA AA
TTA AAC AAG TGG GGC AAA GG
AGT AAC CGC GAT GAA GTG CT
CTT TGC ATC CCG TTT CAG TT
CCT GGT GAT GGC ACT CCT AT
GGG TTG TGG ATC CAG AGA GA
CCA GGA CTT TTG GCC ATT TA
TTC TCT TCT TGG GCT TTG GA
GTC ACT GCT TCG GGT ACC AT
GAT GCA TCC AGT TTC CCA GT
AAC ATC CCC TTT GAC TGC TG
AAG TCC ATG CCC CCT CTA CT
CCT TGG ACT GTG GAA GGT GT
ACC TGC CAG GCT TCA TTC TA
NO and ROS
GGC GCA GGT AAA ACC AAA TA
GCT TTC CTG AAG GGC TTC TT
GTC AGT GAC GAT GAG GCT GA
GTG ATG TCG TCA GGG TTC CT
CGA CGT CTC AGA CCT TCT CC
CCA CGA CTT ACA GCC CAT TT
TGT TGT TGG GCA ACT GAA AA
TTA AAC AAG TGG GGC AAA GG
TTC CCC ACA AGA GCA AAA TC
GCA GGA AAA AGC TGC CTA TG
CGA TGG AGC TTT ACC GAC TC
ATG CAT TTC CCA AAA GCA AG
GGA GGC TCT GTC AGG AGT TG
TGA CTG ATC CCC TGT CCT TC
Cytokine and chemokine
AGT AAC CGC GAT GAA GTG CT
CTT TGC ATC CCG TTT CAG TT
CGC TAC CAG AAC CTG AGG AG
CGG ACG ACA GGA GGT ACA TT
GCC TTC ACG GAA TGT GAT TT
TAG GGG CTA AGA AAC GCA GA
ATG GGT GTT TCA GCC TTC AC
CTG GAC CCA CAT TCT CGT CT
TGA ACC AAA GTG CTG TTC TTA TTT
ACG ATG GAC TTG CAG GAA TC
CCA TGG CTC TCA CTA GCA CA
GCA GGC TGG AGC TCA ATT AC
GGA GGC TCT GTC AGG AGT TG
TGA CTG ATC CCC TGT CCT TC
GGG CGA AAC TTA GCC TCT CT
GGT TGA GGT CAA ACC CAG AA
AAG GGC AAT TTA TGG CAC AG
CCG CTG GTT TCT CCT TAC AA
CTG CAG GAG ACC TCA CAC AA
TCC TCA TTT TCC CTG GAT TG
TTG CAG TGA TTT GCT TTT GC
CAG ACT CGT TGG AGT CGT CA
GCT GCT TTG CCT ACA TTT CC
TCA GGT TCC AGA TGC CCT AC
CCC ATC CAG AAG CTG AAG AG
GCT TGG GTT TTC TTG TCC AG
Statistical analysis was performed using the Statistical Product and Services Solutions (SPSS) package, version 10.0 (SPSS, Cary, NC, USA). Student’s t-test was used. A P-value < 0.05 was considered to be statistically significant. The data from the infected dogs were compared with those from the uninfected dogs.
Morphology of isolated canine H3N2 influenza virus
Clinical signs, viral titers, and aerosol transmission of canine H3N2 influenza virus in dogs
We tested the possibility of aerosol transmission of canine H3N2 influenza virus among dogs to determine whether the avian origin canine H3N2 influenza virus is well adapted in dogs. When the cage containing the infected dogs was placed 90.0 cm apart from the cage containing naïve dogs, the viruses were detected in the nasal swabs of the naïve dogs 3 and 5 days after transmission (Figure 2E), suggesting that canine H3N2 influenza virus has an effective transmission among dogs via aerosols.
Gross pathology, histopathology, and immunohistochemistry of the lungs of infected dogs
Analysis of gene transcription induced in the lungs of infected dogs
We think that the immune responses elicited in the dogs infected with canine H3N2 influenza virus may also contribute to the pathogenesis of influenza.
Dogs live in close contact with humans. We characterized the pathogenicity of the avian origin H3N2 influenza virus in dogs. The infected dogs suffered from severe clinical signs, such as high fever, reduced body weight, and pneumonia. The canine H3N2 influenza virus could be efficiently transmitted via aerosols from the infected dogs to naïve dogs. Analysis of transcription induction in the lungs of infected dogs showed that a variety of genes related to innate and adaptive immunity and cell death were up-regulated.
Our data show that dogs infected with a recently isolated canine H3N2 influenza virus suffered from high fever and severe interstitial pneumonia. The previous study using 2007 canine H3N2 influenza virus also showed that the infected dogs suffered from high fever and the interstitial pneumonia and bronchioalveolitis .
Our results suggest that a canine H3N2 influenza virus is well adapted in dogs since this virus could efficiently transmit itself through aerosols. The aerosol transmission studies were performed to evaluate the potential transmission of influenza virus among humans using an animal model [30–34]. The transmission efficiency of avian origin H5N1 influenza virus in ferrets has been previously studied [30, 31]. The reassortant H5HA/H1N1 virus containing H5 HA with four mutations and the remaining 7 genes from the 2009 pandemic H1N1 influenza virus could transmit among ferrets via droplets, and did not cause severe clinical signs such as death in the transmitted ferret . Another study on the aerosol transmission of avian origin H5N1 influenza virus suggested that the subsequent serial passages of an H5N1 influenza virus in ferrets could result in airborne transmission among ferrets. These transmissible H5N1 viruses contained four amino acid substitutions in the receptor-binding amino acids of HA, and one in the polymerase complex protein basic polymerase 2 . The transmission study in the ferrets carrying avian origin H9N2 influenza virus, which is a potential threat to humans, showed that the H9N2 influenza virus could be transmitted by direct contact with the ferrets, but it could not be transmitted to naïve ferrets via aerosols . The swine-originated, pandemic H1N1 influenza virus of 2009 was easily transmissible among ferrets [33, 34]. When the ferrets were infected with the pandemic H1N1 influenza virus or the seasonal H1N1 influenza virus, both viruses were found to be efficiently transmissible among ferrets via aerosols . In addition, the 2009 pandemic H1N1 influenza virus was transmitted among ferrets via contact and respiratory droplet exposure, before the earliest clinical sign of fever was detected .
Extensive transcriptional genomic analysis revealed that genes related to innate and adaptive immunity, inflammation, and apoptosis were highly active in the lungs of the dogs infected with canine H3N2 influenza virus, more than the lungs of PBS mock-infected dogs. The resulting immune responses of the dogs may also be responsible for contributing to the pathogenicity of the virus. Previous global transcriptional analysis studies also attributed the immune responses, including inflammation and apoptosis, to the enhanced pathogenicity of influenza viruses in animals [35–37]. A global transcriptional analysis was performed in macaques infected with the 1918 pandemic H1N1 or the avian origin H5N1 influenza virus . Specific groups of genes related to inflammation and cell death were elevated in the bronchial tissues of macaques infected with the 1918 pandemic H1N1 influenza virus, but were downregulated by the avian origin H5N1 influenza virus . When mice were infected with the 1918 pandemic H1N1 influenza virus, the genes associated with pro-inflammatory and cell death pathways were seen to be upregulated 24 h after infection, and remained so until death at 5 days . Extensive transcriptional genomic profiles in the lungs of the ferrets showed that the interferon-response genes were more highly expressed in the avian origin H5N1-infected ferret lungs than in the lungs of the ferrets infected with the H3N2 influenza virus, and strong CXCL10 gene expression was induced in the lungs of the ferrets infected with the avian origin H5N1 influenza virus .
Our data show that the gene expression in the lungs of dogs infected with canine H3N2 influenza virus was a little different between data of microarray and qPCR even though the overall patterns are related. The discrepancy between both methods may be due to the inherent pitfall of both methods that may influence the data obtained from each method as described in the previous studies [38–40].
In conclusion, our data suggest that the canine H3N2 influenza virus may be well adapted to infecting dogs, and that the pathogenesis of dogs infected with the canine H3N2 influenza virus may be due to the elevated expression of genes associated with immune responses and cell death in the lungs.
This research was in part supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A2A2A01002533). A staff in Editage edited this manuscript.
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