Canine Influenza Virus Infection Research Articles

Canine Interferon-Inducible Transmembrane Protein Is a Host Restriction Factor That Potently Inhibits Replication of Emerging Canine Influenza Virus.

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro. An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host’s defense against virus infection.

Antiviral Activity of Canine RIG-I against Canine Influenza Virus and Interactions between Canine RIG-I and CIV.

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

PREVALENCE AND ASSOCIATED RISK FACTORS OF CANINE PARVOVIRUS AND CANINE INFLUENZA VIRUS INFECTIONS IN PET DOGS IN DHAKA DISTRICT OF BANGLADESH

Background: There are approximately 1.6 million dogs in Bangladesh and almost 83% of these dogs live on the street and accordingly 17.0% dog population are kept as pet mostly in the metropolitan cities with major population in Dhaka, Chottogram and Sylhet in Bangladesh. Some promiscuous research findings on Canine parvovirus enteritis (PVE) and Canine influenza virus (CIV) have been reported in inland literature. Objective: To determine the prevalence and associated risk factors of canine parvovirus and canine influenza virus infections in dogs supported with brief review for future direction of research and prevention Materials and Methods: A cross sectional study was conducted on total of 173 pet dogs for the prevalence of CIV and 70 dogs for CPV infections of different breed, age and gender by collecting nasal swab samples for CIV and rectal swabs for CPV infection. Each of the collected nasal swabs was tested by RapiGen Canine influenza Ag test kit and rectal swab samples with RapiGen Canine parvovirus Ag test kit (RapiGen INC., South Korea, 2012). Chi-square test was used detect the significance of risk factors of the infections in dogs. Results: All the 173 nasal swabs of pet dogs collected from different thanas of the Dhaka district showed negative with RapiGen CIV Ag test kit test. Out of four published reports on the prevalence of CIV infection in dogs, of which two reports showed 10.71 to 13.33% prevalence rate of CIV whereas two reports (including this one) showed negative result with the same test. An overall 7.14% prevalence of CPV infection in pet dogs was recorded in this study. The prevalence of CPV in relation to breed was found 22.22% in German shepherd and 2.86% in Labrador whereas local, Bull mastiff and Samoyed breeds found negative for CPV infection. The higher prevalence of CPV infection was recorded in puppies up to six months of age (14.81%) than in growing dogs aged between >6 to 12 months (7.14%) whereas adult (>1 to 2 years) and older (> 2 years) dogs found negative to this infection. Comparatively higher prevalence of CPV infection was detected in male (8.33%) than in female (5.88%) dogs. No CPV infection was recorded in vaccinated dogs, whereas 19.23% unvaccinated dogs affected with this infection. All the rectal swab samples of apparently healthy dogs (no sign of diarrhea) showed negative to CPV infection, whereas 25.0% dogs with diarrhea sign found positive to CPV infection. Review of inland literature reveals that out of nine articles published on CPV infection of which RapiGen CPV Ag test kit has been used in four, PCR in one and clinical method of diagnosis in four articles, whereas only RapiGen CIV Ag test kit has been used for the diagnosis of CIV infection. Conclusion: The prevalence of CPE associated with diarrhea in 7.14% pet dogs has been recognized in this investigation with supports of earlier reports whereas the prevalence of CIV in pet dogs varied widely from negative to 13.33% prevalence in dogs. Age and vaccination of dogs have been recognized as primary risk factors which should be considered in planning a control program whereas others factors like breeds, season, geographical areas can be considered as secondary risk factors varied widely in different reports and countries. Comparative evaluation of different diagnostic tests to find out the ‘gold standard’ and vaccination against CPI in puppies may be suggested to control this disease in dogs. Keywords: Prevalence, Risk factors, CIV, CPV, Dogs, Breeds, Dhaka district, RapiGen Ag test kit, Brief review

Cellular microRNAs influence replication of H3N2 canine influenza virus in infected cells

MicroRNAs (miRNAs) are known to play important regulatory roles in host-virus interactions. Avian-origin H3N2 canine influenza virus (CIV) has emerged as the most prevalent subtype among dogs in Asia since 2007. To evaluate the roles of host miRNAs in H3N2 CIV infection, here, miRNA profiles obtained from primary canine bronchiolar epithelial cells (CBECs) and canine alveolar macrophages (CAMCs) were compared between infected and mock-infected cells with the H3N2 CIV JS/10. It was found that the expressions of cfa-miR-125b and cfa-miR-151, which have been reported to be associated with innate immunity and inflammatory response, were significantly decreased in CIV-infected canine primary cells. Bioinformatics prediction indicated that 5′ seed regions of the two miRNAs are partially complementary to the mRNAs of nucleoprotein (NP) and non-structural protein 1 (NS1) of JS/10. As determined by virus titration, quantitative real-time PCR (qRT-PCR) and western blotting, overexpression of the two miRNAs inhibited CIV replication in cell culture, while their inhibition facilitated this replication, suggesting that the two miRNAs could act as negative regulators of CIV replication. Our findings support the notion that some cellular miRNAs can influence the outcome of virus infection, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of H3N2 CIV.

Phosphoproteomics to Characterize Host Response During H3N2 Canine Influenza Virus Infection of Dog Lung.

Avian-origin H3N2 canine influenza viruses (CIVs) cause severe contagious respiratory disease in dogs, and quickly adapt to new environments. To further understand the mechanism of virus infection and host-virus interactions, we characterized the complete phosphoproteome of dogs infected with H3N2 CIV. Nine-week-old Beagle dogs were inoculated intranasally with 106 EID50 of A/canine/Guangdong/04/2014 (H3N2) virus. Lung sections were harvested at 5 days post-inoculation (dpi) and processed for global and quantitative analysis of differentially expressed phosphoproteins. A total of 1,235 differentially expressed phosphorylated proteins were identified in the dog lung after H3N2 CIV infection, and 3,016 modification sites were identified among all differentially expressed proteins. We then performed an enrichment analysis of functional annotations using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) database analyses to predict the functions of the identified differential phosphoproteins. Our data indicate that H3N2 CIV infection causes dramatic changes in the host protein phosphorylation of dog lungs. To our knowledge, this is the first study to assess the effect of H3N2 CIV infection on the phosphoproteome of beagles. These data provide novel insights into H3N2-CIV-triggered regulatory phosphorylation circuits and signaling networks and may improve our understanding of the mechanisms underlying CIV pathogenesis in dogs.

Metabolic Profiles in Madin-Darby Canine Kidney Cell Lines Infected with H3N2 Canine Influenza Viruses.

Virus replication and host cell growth require host cell metabolic networks to provide energy and precursors for the synthesis of macromolecules. The aim of this study was to investigate the most direct changes in energy metabolism and small-molecule metabolism of Madin-Darby canine kidney (MDCK) cells infected with H3N2 canine influenza virus (CIV) and to determine whether small metabolites contribute to the pathogenesis of CIV. To study the metabolomics of MDCK cells infected with H3N2 CIV, we used liquid chromatography-tandem mass spectrometry combined with multivariate statistical analysis. The results showed that 798 positive ions were detected, among which 33 were upregulated and 11 were downregulated, and 406 negative ions were detected, among which 33 were upregulated and 9 were downregulated. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, we found that these differentially expressed molecules were mainly concentrated in the steroid hormone biosynthesis, amino sugar and nucleotide sugar metabolism, sphingolipid metabolism, vitamin B6 metabolism, cysteine and methionine metabolism, vitamin digestion and absorption, arginine and proline metabolism, biosynthesis of amino acids, and folate biosynthesis metabolic pathways. These pathways are involved in energy metabolism and nucleic acid and protein synthesis, which are essential for virus replication. Our experimental data suggest that H3N2 CIV infection reconstitutes/influences cellular metabolic processes, which in turn may contribute to viral replication. These findings are important for the development of enzyme inhibitors or metabolites for the identification of antiviral drugs. In addition, understanding the metabolic interaction between CIV and host cells is also very important for the complex pathogenicity of CIV, providing certain guidance for the treatment of canine influenza.

Role of CARD Region of MDA5 Gene in Canine Influenza Virus Infection

MDA5 belongs to the RIG-I-like receptor family, which is involved in innate immunity. During viral infection, MDA5 generates an antiviral response by recognizing the ligand to activate interferon. However, the role and mechanism of MDA5 in canine influenza virus (CIV) infection are unclear. To understand the mechanism of canine MDA5-mediated innate immunity during CIV infection, we detected the distribution of MDA5 in beagles, and the structural prediction showed that MDA5 was mainly composed of a CARD domain, RD domain, and DExD/H helix structure. Moreover, we found that MDA5 inhibits CIV replication. Furthermore, in the dual luciferase assay, we revealed that the CARD region of MDA5 strongly activated the IFN-β promoter and mainly transmitted signals through the CARD region. Overexpression of the CARD region of MDA5 revealed that the MDA5-mediated signaling pathway could transmit signals by activating the IRF3/NF-κB and IRF3 promoters, promoting the expression of antiviral proteins and cytokine release, thereby inhibiting CIV replication. Upon silencing of MDA5, cytokine production decreased, while the replication ability of CIV was increased. Thus, this study revealed a novel mechanism by which MDA5 mediated CIV infection and provided new avenues for the development of antiviral strategies.

Emergence and Containment of Canine Influenza Virus A(H3N2), Ontario, Canada, 2017-2018.

Canine influenza virus (CIV) A(H3N2) was identified in 104 dogs in Ontario, Canada, during December 28, 2017–October 30, 2018, in distinct epidemiologic clusters. High morbidity rates occurred within groups of dogs, and kennels and a veterinary clinic were identified as foci of infection. Death attributable to CIV infection occurred in 2 (2%) of 104 diagnosed cases. A combination of testing of suspected cases, contact tracing and testing, and 28-day isolation of infected dogs was used, and CIV transmission was contained in each outbreak. Dogs recently imported from Asia were implicated as the source of infection. CIV H3N2 spread rapidly within groups in this immunologically naive population; however, containment measures were apparently effective, demonstrating the potential value of prompt diagnosis and implementation of CIV control measures.

H3N2 canine influenza virus and Enterococcus faecalis coinfection in dogs in China

BackgroundIn May 2017, 17 dogs in a German Shepherd breeding kennel in northern China developed respiratory clinical signs. The owner treated the dogs with an intravenous injection of Shuang-Huang-lian, a traditional Chinese medicine, and azithromycin. The respiratory signs improved 3 days post-treatment, however, cysts were observed in the necks of eight dogs, and three of them died in the following 2 days.Case presentationQuantitative real-time PCR was used to detect canine influenza virus (CIV). All of the dogs in this kennel were positive and the remaining 14 dogs had seroconverted. Two of the dogs were taken to the China Agricultural University Veterinary Teaching Hospital for further examination. Two strains of influenza virus (A/canine/Beijing/0512–133/2017 and A/canine/Beijing/0512–137/2017) isolated from the nasal swabs of these dogs were sequenced and identified as avian-origin H3N2 CIV. For the two dogs admitted to the hospital, hematology showed mild inflammation and radiograph results indicated pneumonia. Cyst fluid was plated for bacterial culture and bacterial 16 s rRNA gene PCR was performed, followed by Sanger sequencing. The results indicated an Enterococcus faecalis infection. Antimicrobial susceptibility tests were performed and dogs were treated with enrofloxacin. All 14 remaining dogs recovered within 16 days.ConclusionsCoinfection of H3N2 CIV and Enterococcus faecalis was detected in dogs, which has not been reported previously. Our results highlight that CIV infection might promote the secondary infection of opportunistic bacteria and cause more severe and complicated clinical outcomes.

Serological evidence of H3N2 canine influenza virus infection among horses with dog exposure.

Currently, Canine influenza virus (CIV) H3N2 is continuously circulating in dog populations in China, Korea, and the United States (US). Both influenza SA-α-2,3-Gal and SA-α-2,6-Gal receptors have been observed in the respiratory tracts of both horses and dogs. Hence, the increasing number of CIV H3N2 cases in the world indicates a potential risk for transspecies transmission to horses with dog exposure. Here, a seroepidemiological survey of CIV H3N2 infections in horses was conducted using hemagglutination inhibition (HI), microneutralization (MN) and the chicken embryo neutralization test (CENT). From April 2014 to November 2016, 399 sera from race horses were collected in Guangzhou, Dongguan, Huizhou, and Shenzhen in China. Nine specimens (2.2%, 9/399) were positive for CIV H3N2 with HI titers≥1:20, MN titers≥1:80 and CENT titers≥1:80. Furthermore, these positive horses showed significant correlation with dog exposure, and some dogs (20%, 3/15) from the same riding clubs as the positive horses also possessed antibodies against CIV H3N2. This study is the first to provide seroepidemiological evidence of CIV H3N2 infection in horses with exposure to dogs. Based on these findings, continuous serological and virological surveillance of CIV H3N2 infection among horses is imperative, and further animal experiments should be performed.

Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

Comparative pathogenesis of H3N2 canine influenza virus in beagle dogs challenged by intranasal and intratracheal inoculation

As important companion animals, dogs may serve as intermediate hosts for transmitting influenza virus to humans. However, knowledge regarding H3N2 canine influenza virus (CIV) pathogenicity is not comprehensive, which directly affects the animal models of pathogenicity in H3N2 CIV vaccine research. Here, to assess H3N2 CIV pathogenicity, we utilized 30 ten-week-old purpose-bred beagles intratracheally or intranasally inoculated with 106 50% egg-infectious dose. Intratracheal inoculation was more virulent to dogs than intranasal inoculation as shown by lung pathology score, histopathological changes, clinical symptoms, and body temperature. More intense virus replication was observed in the upper and lower respiratory tracts by intratracheal than intranasal inoculation according to nasal swabs, various organ virus titers, and antigen expression. These results may enhance the H3N2 CIV infection model, providing a more complete experimental basis for studying intrinsic H3N2 CIV pathogenic mechanism, and also serving a reference role for CIV prevention and treatment.

Outcomes of and risk factors for presumed canine H3N2 influenza virus infection in a metropolitan outbreak.

OBJECTIVE To determine clinical signs, case fatality rate, and factors associated with positive results of PCR testing for canine influenza virus (CIV) in dogs during an H3N2 CIV outbreak in the Atlanta area. DESIGN Cross-sectional study. ANIMALS 220 dogs with a nasal swab specimen submitted to an Atlanta-area diagnostic laboratory between May 1 and July 2, 2015, for PCR assay detection of CIV specifically or CIV and 5 other respiratory pathogens. PROCEDURES Veterinarians of tested dogs were surveyed by various means to collect information regarding clinical signs, survival status at the time of survey completion, vaccination history (≤ 12 months prior to testing), and travel history (≤ 2 months prior to testing). Data were compared between CIV-positive and CIV-negative dogs. RESULTS Surveys for 120 (55%) dogs were completed. Forty (33%) of these dogs had positive results of CIV testing. No significant differences were identified between CIV-positive and CIV-negative dogs regarding breed, sex, reproductive status, duration of clinical signs prior to testing, other dogs in the household, or travel history. When other factors were controlled for, CIV-positive dogs were more likely to be adult (> 1 year of age) than juvenile (≤ 1 year of age) and to be inappetent. Only 1 (3%) CIV-positive dog died during the study period (shortly after it was evaluated because of respiratory signs). CONCLUSIONS AND CLINICAL RELEVANCE From May 1 to July 2, 2015, the reported clinical signs of dogs tested during the Georgia H3N2 CIV outbreak were similar to those reported for dogs with H3N8 CIV infection, and the case fatality rate was low.

Integrated Lung and Tracheal mRNA-Seq and miRNA-Seq Analysis of Dogs with an Avian-Like H5N1 Canine Influenza Virus Infection.

Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID50) of 106, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.

Serological evidence of canine influenza virus infection in shelter dogs in Turkey

Influenza virus infection is an important disease which occurs in humans and a variety of animals. Because of the wide host adaptation and segmented genome, there is always the possibility of mutations and interspecies transmission of the influenza virus. Our study is the first to draw attention to canine influenza infection in Turkey. For this purpose, 208 sera and swab samples were collected from dogs with respiratory and nonrespiratory signs in various seasons. Out of the 208 dogs, 94 (45.2%) were male and 114 (54.8%) were female; the average age was 4.7 years. A total of 208 sera samples were tested for the presence of canine influenza virusspecific antibodies by the indirect enzyme-linked immunosorbent assay. The seroprevalence of canine influenza virus infection was 11/208 (5.8%). With regard to seasonal distribution, the highest rate of seropositivity was detected in spring, and the lowest in summer. Molecular detection of the canine influenza virus from nasal swab samples was done by reverse transcriptase polymerase chain reaction using specific universal primers for the hemagglutinin gene. Influenza virus nucleic acid could not be detected by reverse transcriptase polymerase chain reaction. In this study, we revealed for the first time the existence of the canine influenza virus in Turkey. Although the seroprevalence was relatively low, it would be useful to investigate the canine influenza virus on a large scale and among dogs with infectious respiratory disease in the Turkish dog population..

Prevalence of canine parvo virus and canine influenza virus infection in dogs in Dhaka, Mymensingh, Feni and Chittagong districts of Bangladesh

Canine parvovirus (CPV) and canine influenza virus (CIV) are highly contagious virus infecting members of the canine family including dogs, coyotes, foxes and wolves. The research work was carried out to determine the prevalence of CPV and CIV in dogs (stray and pet dogs) in Dhaka, Mymensingh, Feni and Chittagong Districts by using Rapigen CPV and CIV Ag test kit. Fecal samples were collected from 50 dogs of different age and sex from different spots of Dhaka and Chittagong District. Nasal fluid samples were collected from 50 dogs of different age and sex from different spots. The samples were tested with Rapigen canine parvovirus Ag test kit and Rapigen canine influenza Ag test kit. Overall prevalence of CPV in pet dogs was 22% and in stray dogs was 30%. The prevalence of canine parvovirus in relation to age was gradually decreasing with higher age group and the prevalence was 28% in 1-6 months age, 16.66% in 7-12 months age group 11.11%o ver 18 months age group. The prevalence of canine parvovirus was significantly higher in male (24.13%) than that in female (19.04%). All samples showed negative test result for canine influenza virus. Prevalence of canine influenza virus infection were 00.00% in all age groups and sex. Overall prevalence of CIV in all pet and stray dogs were 00.00%.Asian J. Med. Biol. Res. March 2016, 2(1): 138-142

Identification of the IFN-β response in H3N2 canine influenza virus infection.

Canine influenza viruses (CIVs) circulate continuously in the dog population, providing opportunities for exposure to humans and other species. Although the dog genome has been sequenced, innate immunity in dogs is not well characterized, which limits the understanding of H3N2 canine influenza virus pathogenesis. Equally, how this virus evades the canine host innate immune response to successfully establish infection remains unclear. To analyse the IFN-β response to CIV infection in Madin-Darby canine kidney cells, the canine IFN-β promoter sequence and its positive regulatory domain motifs were first cloned and identified using a luciferase reporter system. Next, we found that infection with the CIV strain GD/12 blocked the IFN-β response primarily by inhibiting the NF-κB and IFN regulatory factor 3 (IRF3) signalling pathways. Expression of GD/12 non-structural protein 1 alone was sufficient to inhibit Sendai virus-induced NF-κB and IRF3 activation by suppressing p65 and IRF3 phosphorylation, suggesting the important role of this protein in the CIV-mediated inhibition of the IFN-β response. These results suggest that inhibition of the IFN-β signalling pathway may have played a role in CIV establishment and spread in dog populations.

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus.

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-β were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-β mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-β mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-β than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.

Monoclonal antibody specific to HA2 glycopeptide protects mice from H3N2 influenza virus infection.

Canine influenza virus (CIV) subtype H3N2 is a newly identified, highly contagious respiratory pathogen that causes cough, pneumonia and other respiratory symptoms in dogs. Data indicate that the virus is responsible for recent clinical cases of dog disease in China. However, therapeutic options for this disease are very limited. In this study, seven monoclonal antibodies (mAbs) against CIV JS/10 (an H3N2 subtype virus) were produced and characterized. Among them, mAb D7, which is specific for the HA2 glycopeptide (gp), induced the highest neutralization titers. The protection provided by mAb D7 was evaluated in BALB/c mice challenged with homologous or heterologous strains of H3N2 influenza virus, including two strains of CIV and one strain of swine influenza virus (SIV). The data show that mAb D7 protected the mice from infection with the three viral strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. Moreover, the levels of IFN-γ and TNF-α in the lungs, as detected by ELISA, were reduced in the infected mice treated with the mAb D7 compared with those without mAb D7 treatment. Thus, our findings demonstrate, for the first time, that a mAb could reduce the release of IFN-γ and TNF-α associated with tissue damage by CIV infection and that the mAb might be of great therapeutic value for CIV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0146-7) contains supplementary material, which is available to authorized users.

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets, China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.