Canine H3N2 Research Articles

Utilizing machine learning and hemagglutinin sequences to identify likely hosts of influenza H3Nx viruses

Influenza is a disease that represents both a public health and agricultural risk with pandemic potential. Among the subtypes of influenza A virus, H3 influenza virus can infect many avian and mammalian species and is therefore a virus of interest to human and veterinary public health. The primary goal of this study was to train and validate classifiers for the identification of the most likely host species using the hemagglutinin gene segment of H3 viruses. A five-step process was implemented, which included training four machine learning classifiers, testing the classifiers on the validation dataset, and further exploration of the best-performing model on three additional datasets. The gradient boosting machine classifier showed the highest host-classification accuracy with a 98.0 % (95 % CI [97.01, 98.73]) correct classification rate on an independent validation dataset. The classifications were further analyzed using the predicted probability score which highlighted sequences of particular interest. These sequences were both correctly and incorrectly classified sequences that showed considerable predicted probability for multiple hosts. This showed the potential of using these classifiers for rapid sequence classification and highlighting sequences of interest. Additionally, the classifiers were tested on a separate swine dataset composed of H3N2 sequences from 1998 to 2003 from the United States of America, and a separate canine dataset composed of canine H3N2 sequences of avian origin. These two datasets were utilized to look at the applications of predicted probability and host convergence over time. Lastly, the classifiers were used on an independent dataset of environmental sequences to explore the host identification of environmental sequences. The results of these classifiers show the potential for machine learning to be used as a host identification technique for viruses of unknown origin on a species-specific level.

Development of Live Vaccine Candidates for Canine Influenza H3N2 Using Naturally Truncated NS1 Gene

The NS1 influenza protein of influenza A virus is a viral nonstructural protein encoded by the NS gene segment that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced protein. We isolated an equine influenza virus with a naturally truncated NS1 gene in our previous study. In this current research, we inserted this partially truncated NS gene into the H3N2 canine influenza virus using reverse genetics to develop a live attenuated vaccine strain. To evaluate whether the developed strain is suitable as a live vaccine candidate, we compared its replication kinetics with wild-type virus in MDCK cells and specific pathogen-free eggs. Additionally, we investigated host antiviral gene expression, viral replication in the respiratory system, and associated lung tissue damage in mice experiments. To confirm the efficacy of the vaccine candidate, we evaluated the immunogenicity and protectivity of the developed vaccine strain against canine influenza H3N2, compared with a commercial inactivated vaccine. Through these experiments, it was confirmed that the naturally truncated NS1 inserted virus has sufficient potential as a live vaccine candidate, and we hopefully expect that this study would make a great contribution to the development of a live vaccine for canine influenza H3N2.

Transmission and pathogenicity of canine H3N2 influenza virus in dog and guinea pig models

BackgroundInfluenza A virus causes respiratory disease in many animal species as well as in humans. Due to the high human-animal interface, the monitoring of canine influenza in dogs and the study of the transmission and pathogenicity of canine influenza in animals are important.MethodsEight-week-old beagle dogs (Canis lupus familaris) (n = 13) were used for the intraspecies transmission model. The dogs were inoculated intranasally with 1 ml of 106 EID50 per ml of canine H3N2 influenza virus (A/canine/Thailand/CU-DC5299/2012) (CIV-H3N2). In addition, 4-week-old guinea pigs (Cavia porcellus) (n = 20) were used for the interspecies transmission model. The guinea pigs were inoculated intranasally with 300 µl of 106 EID50 per ml of CIV-H3N2.ResultsFor the Thai CIV-H3N2 challenged in the dog model, the incoculated and direct contact dogs developed respiratory signs at 2 dpi. The dogs shed the virus in the respiratory tract at 1 dpi and developed an H3-specific antibody against the virus at 10 dpi. Lung congestion and histopathological changes in the lung were observed. For the Thai CIV-H3N2 challenge in the guinea pig model, the incoculated, direct contact and aerosol-exposed guinea pigs developed fever at 1–2 dpi. The guinea pigs shed virus in the respiratory tract at 2 dpi and developed an H3-specific antibody against the virus at 7 dpi. Mild histopathological changes in the lung were observed.ConclusionThe result of this study demonstrated evidence of intraspecies and interspecies transmission of CIV-H3N2 in a mammalian model.

Emergence and Evolution of Novel Canine-Avian Reassortant H3N2 Influenza A Viruses in Duck in Leizhou Peninsula, China.

Avian-to-mammal transmission and mammalian adaptation of avian influenza virus (AIV) are threats to public health and of great concern. The H3 subtype of influenza virus has low pathogenicity and is widely distributed in humans, canines, equines and avians. In 2018–2019, we isolated six H3N2 subtype influenza viruses from 329 samples acquired from ducks on the Leizhou Peninsula, China, as part of an ongoing virus surveillance program. All viruses were analyzed by whole-genome sequencing with subsequent genetic comparison and phylogenetic analysis. Phylogenetic analysis demonstrated that reassortment of these viruses has occurred among different hosts and subtypes. Some of the H3 AIV isolates have similar genes as subtypes H5 and H7 of highly pathogenic avian influenza viruses (HPAIVs). Most importantly, one strain of H3N2 virus is a novel reassortant influenza virus containing HA and PB2 segments from canine H3N2 virus. The time of most recent common ancestor (tMRCA) data indicated that this reassortant H3N2 virus might have emerged in 2011–2018. The findings suggest that the viruses studied here have undergone multiple reassortment events. Our results provide a framework for understanding the molecular basis of host-range shifts of influenza viruses and we should pay more attention to canine which lived with avian together.

Characterization of Canine Influenza Virus A (H3N2) Circulating in Dogs in China from 2016 to 2018.

Avian H3N2 influenza virus follows cross-host transmission and has spread among dogs in Asia since 2005. After 2015–2016, a new H3N2 subtype canine influenza epidemic occurred in dogs in North America and Asia. The disease prevalence was assessed by virological and serological surveillance in dogs in China. Herein, five H3N2 canine influenza virus (CIV) strains were isolated from 1185 Chinese canine respiratory disease samples in 2017–2018; these strains were on the evolutionary branch of the North American CIVs after 2016 and genetically far from the classical canine H3N2 strain discovered in China before 2016. Serological surveillance showed an HI antibody positive rate of 6.68%. H3N2 was prevalent in the coastal areas and northeastern regions of China. In 2018, it became the primary epidemic strain in the country. The QK01 strain of H3N2 showed high efficiency in transmission among dogs through respiratory droplets. Nevertheless, the virus only replicated in the upper respiratory tract and exhibited low pathogenicity in mice. Furthermore, highly efficient transmission by direct contact other than respiratory droplet transmission was found in a guinea pig model. The low-level replication in avian species other than ducks could not facilitate contact and airborne transmission in chickens. The current results indicated that a novel H3N2 virus has become a predominant epidemic strain in dogs in China since 2016 and acquired highly efficient transmissibility but could not be replicated in avian species. Thus, further monitoring is required for designing optimal immunoprophylactic tools for dogs and estimating the zoonotic risk of CIV in China.

Triple reassortment increases compatibility among viral ribonucleoprotein genes of contemporary avian and human influenza A viruses.

Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.

Influenza Virus Infections in Cats.

In the past, cats were considered resistant to influenza. Today, we know that they are susceptible to some influenza A viruses (IAVs) originating in other species. Usually, the outcome is only subclinical infection or a mild fever. However, outbreaks of feline disease caused by canine H3N2 IAV with fever, tachypnoea, sneezing, coughing, dyspnoea and lethargy are occasionally noted in shelters. In one such outbreak, the morbidity rate was 100% and the mortality rate was 40%. Recently, avian H7N2 IAV infection occurred in cats in some shelters in the USA, inducing mostly mild respiratory disease. Furthermore, cats are susceptible to experimental infection with the human H3N2 IAV that caused the pandemic in 1968. Several studies indicated that cats worldwide could be infected by H1N1 IAV during the subsequent human pandemic in 2009. In one shelter, severe cases with fatalities were noted. Finally, the highly pathogenic avian H5N1 IAV can induce a severe, fatal disease in cats, and can spread via cat-to-cat contact. In this review, the Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European countries, summarises current data regarding the aetiology, epidemiology, pathogenesis, clinical picture, diagnostics, and control of feline IAV infections, as well as the zoonotic risks.

Sequence dynamics of three influenza A virus strains grown in different MDCK cell lines, including those expressing different sialic acid receptors.

Viruses are often cultured in cell lines for research and vaccine development, and those often differ from the natural hosts or tissues. Cell lines can also differ in the presence of virus receptors, such as the sialic acid (Sia) receptors used by influenza A viruses (IAV), which can vary in linkage (α2,3- or α2,6-linkage) and form (N-glycolylneuraminic acid [Neu5Gc] or N-acetylneuraminic acid [Neu5Ac]). The selective pressures resulting from passaging viruses in cell types with host-specific variations in viral receptors are still only partially understood. IAV are commonly cultured in MDCK cells which are both derived from canine kidney tubule epithelium and inherently heterogeneous. MDCK cells naturally present Neu5Ac and α2,3-linked Sia forms. Here, we examine natural MDCK variant lineages, as well as engineered variants that synthesize Neu5Gc and/or α2,6-linkages. We determined how viral genetic variation occurred within human H3N2, H1N1 pandemic and canine H3N2 IAV populations when serially passaged in MDCK cell lines that vary in cell type (MDCK-Type I or MDCK-Type II clones) and in Sia display. Deep sequencing of viral genomes showed small numbers of consensus-level mutations, mostly within the hemagglutinin (HA) gene. Both human IAV showed variants in the HA stem and the HA receptor-binding site of populations passaged in cells displaying Neu5Gc. Canine H3N2 showed variants near the receptor-binding site when passaged in cells displaying Neu5Gc or α2,6-linkages. Viruses replicated to low titres in MDCK-Type II cells, suggesting that not all cell types in heterogeneous MDCK cell populations are equally permissive to infection.

Patchless administration of canine influenza vaccine on dog’s ear using insertion-responsive microneedles (IRMN) without removal of hair and its in vivo efficacy evaluation

Microneedles provide the advantages of convenience and compliance by avoiding the pain and fear of needles that animals often experience. Insertion-responsive microneedles (IRMN) were used for administration to a hairy dog without removing the dog’s hair. Canine H3N2 vaccine was administered with IRMN attached to the dog’s ears ex vivo and the conventional microneedle system (MN) was administered for 15 min to compare puncture performance and delivery efficiency. The vaccine was also administered to compare antibody formation using IRMN with the use of intramuscular injection. The veterinarian observed the behavior of the dog during the course of the administration and compared the response to IRMN with that of intramuscular administration.The tips of IRMN were separated from the base and delivered into the hairy skin successfully. Puncture performance of IRMN were the same as that of coated microneedles (95%), but delivery efficiency of IRMN were 95% compared to less than 1% for coated microneedles. The H3N2 vaccine inoculated into the dog's ears showed the same antibody formation as the intramuscular injection. The dog appeared to be more comfortable with IRMN administration compared to syringe administration.IRMN are the first microneedle system to deliver a canine vaccine successfully into a hairy dog without removal of the dog’s hair. The use of IRMN can provide both convenience and compliance for both the dog and the owner.

Influenza A Virus Infection in Cats and Dogs: A Literature Review in the Light of the "One Health" Concept.

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Constantly evolving and crossing species barrier, the emergence of novel zoonotic pathogens is one of the greatest challenges to global health security. During the last decade, considerable attention has been paid to influenza virus infections in dogs, as two canine H3N8 and H3N2 subtypes caused several outbreaks through the United States and Southern Asia, becoming endemic. Cats, even though less documented in the literature, still appear to be susceptible to many avian influenza infections. While influenza epidemics pose a threat to canine and feline health, the risks to humans are largely unknown. Here, we review most recent knowledge of the epidemiology of influenza A viruses in dogs and cats, existing evidences for the abilities of these species to host, sustain intraspecific transmission, and generate novel flu A lineages through genomic reassortment. Such enhanced understanding suggests a need to reinforce surveillance of the role played by companion animals-human interface, in light of the “One Health” concept and the potential emergence of novel zoonotic viruses.

Data capture and visualization for a canine influenza outbreak — New York City, 2018

ObjectiveThe objectives of this project were to rapidly build and deploy a web-based reporting platform in response to a canine influenza H3N2 outbreak in New York City (NYC) and provide aggregate data back to the veterinary community as an interactive dashboard.IntroductionData-driven decision-making is a cornerstone of public health emergency response; therefore, a highly-configurable and rapidly deployable data capture system with built-in quality assurance (QA; e.g., completeness, standardization) is critical.1 Additionally, to keep key stakeholders informed of developments during an emergency, data need to be shared in a timely and effective manner. Dynamic data visualization is a particularly useful means of sharing data with healthcare providers and the public.2During Spring 2018, detection of canine influenza H3N2 among dogs in NYC caused concern in the veterinary community. Canine influenza is a highly contagious respiratory infection caused by an influenza A virus.3 However, no central database existed in NYC to monitor the outbreak and no single agency was responsible for data capture. Our team at the NYC Department of Health and Mental Hygiene (DOHMH) partnered with the NYC Veterinary Medical Association (VMA) to monitor the canine influenza H3N2 outbreak by building a web-based reporting platform and interactive dashboard.MethodsThe NYC DOHMH built and deployed a web-based reporting platform to aid veterinarians in reporting cases of canine influenza. We leveraged REDCap Cloud, a cloud-based graphical user interface data capture and management software. REDCap Cloud collected information regarding the provider, owner, dog, residence of dog, illness history, and influenza testing. We leveraged REDCap QA functionality in the form of mandatory questions to ensure data completeness. Several different field types — including dropdown menus, mutually exclusive radio buttons, and multi-select check boxes — were used to ensure data standardization. Skip logic was incorporated to guide users through unique sequences of questions based on the answers they entered. Reporting was voluntary.ResultsAfter requirements were gathered, the REDCap web-based reporting platform was rapidly deployed in approximately two business days. Over the course of one week, multiple versions of the dashboard were produced and the final iteration was completed. The entire system was built on server-side software that is available as free or open-source for individual licenses. The dashboard can be found at the following link: http://www.vmanyc.org/canine_influenza_dashboard.html.A total of 28 cases were reported by 6 providers during June–August 2018. All of the 28 cases were reported from 2 of the 5 NYC counties (boroughs); 17/28 (60.7%) were reported from Brooklyn and 11/28 (39.3%) were reported from Manhattan. We were able to collect mostly complete data by leveraging REDCap QA functionality. The reporting facility was listed in all cases, and an owner was listed in all but two cases. All reported cases used a PCR test for the detection of canine influenza H3N2. One reported case indicated polymerase chain reaction (PCR) test results as “not detected” which suggests that one negative case was reported through the system.ConclusionsUsing REDCap Cloud and R, we were able to rapidly build and deploy a web-based reporting platform and dynamic data visualization during an emergency response to an outbreak of canine influenza H3N2. Our system was used by veterinarians to report 28 cases of canine influenza. Future emergency responses for human disease outbreaks will likely benefit from the experience our team gained during our partnership with the NYC VMA.

Vaccination with an inactivated canine influenza H3N2 virus vaccine is safe and elicits an immune response in cats.

ObjectivesThe aim of this study was to evaluate safety and seroconversion when an inactivated H3N2 canine influenza virus (CIV) vaccine was administered to cats.MethodsTwenty 7–8-week-old seronegative cats were randomly assigned to two groups of 10 animals each. Cats in treatment group T01 were subcutaneously administered two doses of an adjuvanted placebo 3 weeks apart to serve as non-immunized controls. Cats in treatment group T02 were subcutaneously administered with two doses of H3N2 CIV vaccine at 3 weeks apart. All animals were actively monitored for 5 days after each injection for local and systemic reactions. Tympanic temperatures were recorded the day before and 5 days after each vaccination. Blood samples for serology were collected prior to each vaccination (days –1 and 20), and 7 and 14 days post-second vaccination.ResultsMinor vocalization was observed in both control and vaccinated animals after the first and second dose administration. The only injection site reaction observed was mild swelling in one control cat, which resolved within 24 h. Transient fevers (39.5–39.7°C) that resolved within 24 h post-injection were observed in both treatment groups (T01 = 3/10 and T02 = 5/10). All vaccinated, but no control, animals successfully seroconverted within 14 days of second vaccination, with H3N2 CIV-specific hemagglutination inhibition (HAI) titers ranging from 32 to 128.Conclusions and relevanceCats vaccinated subcutaneously with an inactivated H3N2 CIV vaccine had similar rates of adverse events post-vaccination as the control group. Increased HAI titers provided evidence of post-vaccination seroconversion with the H3N2 CIV-vaccinated group.

Evidence of pandemic H1N1 influenza exposure in dogs and cats, Thailand: A serological survey.

Influenza A virus causes respiratory disease in both humans and animals. In this study, a survey of influenza A antibodies in domestic dogs and cats was conducted in 47 animal shelters in 19 provinces of Thailand from September 2011 to September 2014. One thousand and eleven serum samples were collected from 932 dogs and 79 cats. Serum samples were tested for influenza A antibodies using a multi-species competitive NP-ELISA and haemagglutination inhibition (HI) assay. The NP-ELISA results showed that 0.97% (9/932) of dogs were positive, but all cat samples were negative. The HI test against pandemic H1N1, human H3N2 and canine H3N2 showed that 0.64% (6/932) and 1.20% (1/79) of dogs and cats were positive, respectively. It is noted that all six serum samples (5 dogs and 1 cat) had antibodies against pandemic H1N1. In summary, a serological survey revealed the evidence of pandemic H1N1 influenza exposure in both dogs and cats in the shelters in Thailand.

Serosurvey for Influenza Virus Subtypes H3N8 and H3N2 Antibodies in Free-Ranging Canids in Pennsylvania, USA.

Canine influenza virus (CIV) subtypes H3N8 and H3N2 are endemic among domestic dog ( Canis lupus familiaris ) populations in the northeastern US. Infection of free-ranging carnivores with influenza virus has been sporadically reported. Generalist mesocarnivores that exploit anthropogenic, peri-urban habitats share a wide interface with domestic dogs that allows for the transmission of infectious disease. To investigate the potential exposure of free-ranging canids to CIV in Pennsylvania, US, serum samples were obtained from freshly killed coyotes ( Canis latrans, n=67), grey foxes ( Urocyon cinereoargenteus, n=8), and red foxes ( Vulpes vulpes, n=5) from 24 counties. Animals were harvested during the January-February 2017 hunting season. We failed to detect antibodies to CIV subtypes H3N2 and H3N8 by using hemagglutination inhibition assays validated for domestic dogs. Results suggest CIV was not endemic in free-ranging canid populations in Pennsylvania or that prevalence was too low to be detected by our limited sample size.

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.

Comparison of the virulence and transmissibility of canine H3N2 influenza viruses and characterization of their canine adaptation factors

Recent canine influenza outbreaks have raised concerns about the generation of pathogenic variants that may pose a threat to public health. Here, we examine avian-like H3N2 canine influenza viruses (CIVs) isolated from 2009 to 2013 in South Korea from dogs. Phylogenetic analysis revealed that these viruses are closely related to strains previously isolated from dogs in Korea and China. However, molecular characterization demonstrated non-synonymous mutations between the canine viruses, particularly in the putative H3 antigenic sites, NA stalk regions, and in the internal genes of the 2012–2013 isolates compared with the 2009 isolate. Animal experiments showed that three representative isolates (A/canine/Korea/AS-01/2009(AS-01/09), A/canine/Korea/AS-05/2012(AS-05/12) and A/canine/Korea/AS-11/2013(AS-11/13), were readily droplet transmitted between dogs, whereas AS-05/12 induced more severe clinical disease and was lethal in dogs compared with AS-01/09. Although all viruses were able to infect ferrets, AS-05/12 consistently yielded higher nasal wash titers and was transmissible to ferrets via airborne droplets. Using reverse genetics, we show that the NA, NP, and M genes of CIV are critical for the adaptation of avian H3N2 viruses, and the resulting reassortant genotypes promote viral growth in dogs in a manner similar to that of the wild-type AS-01/09 virus. Taken together, these results demonstrate that CIVs continuously evolve in dogs thereby allowing them to gain a foothold in mammalian hosts. Importantly, we elucidated the genetic contributions of the NA, NP, and M genes to the adaptability of CIVs derived from the avian H3N2 virus.

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells.

Vaccination is the primary strategy for influenza prevention and control. However, egg-based vaccines, the predominant production platform, have several disadvantages, including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted the development of cell-based vaccines, which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR-based mutagenesis strategy, we identified a Y161F mutation in hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold but also increased its thermostability without changing its original antigenic properties. The vaccine produced from the Y161F mutant fully protected mice against lethal challenge with wild-type A(H1N1)pdm09. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, the introduction of the Y161F mutation into HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses.IMPORTANCE Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one large challenge: high-yield vaccine seeds for production. In this study, we identified a molecular signature, Y161F, in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two cell lines commonly used for influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability but also enhanced its binding affinity for α2,6- and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine.

Two influenza A virus subtypes has been reported in dogs in the last 16 years: the canine influenza viruses (CIV) H3N8 and H3N2 of equine and avian origins, respectively. To date, only inactivated influenza vaccines (IIVs) are available to prevent CIV infections. Here, we report the generation of a recombinant, temperature-sensitive H3N8 CIV as a live-attenuated influenza vaccine (LAIV), which was attenuated in mice and dog tracheal, explants compared to CIV H3N8 wild type. A single dose of H3N8 LACIV showed immunogenicity and protection against a homologous challenge that was better than that conferred with an H3N8 IIV, demonstrating the feasibility of implementing a LAIV approach for the improved control of H3N8 CIV infections in dogs.

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.

Seroprevalence of three influenza A viruses (H1N1, H3N2, and H3N8) in pet dogs presented to a veterinary hospital in Ohio.

The prevalence of canine H3N8 influenza and human H1N1 and H3N2 influenza in dogs in Ohio was estimated by conducting serologic tests on 1,082 canine serum samples. In addition, risk factors, such as health status and age were examined. The prevalences of human H1N1, H3N2, and canine H3N8 influenzas were 4.0%, 2.4%, and 2.3%, respectively. Two samples were seropositive for two subtypes (H1N1 and H3N2; H1N1 and canine influenza virus [CIV] H3N8). Compared to healthy dogs, dogs with respiratory signs were 5.795 times more likely to be seropositive against H1N1 virus (p = 0.042). The prevalence of human flu infection increased with dog age and varied by serum collection month. The commercial enzyme-linked immunosorbent assay used in this study did not detect nucleoprotein-specific antibodies from many hemagglutination inhibition positive sera, which indicates a need for the development and validation of rapid tests for influenza screening in canine populations. In summary, we observed low exposure of dogs to CIV and human influenza viruses in Ohio but identified potential risk factors for consideration in future investigations. Our findings support the need for establishment of reliable diagnostic standards for serologic detection of influenza infection in canine species.