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.

Emergence and Characterization of a Novel Reassortant Canine Influenza Virus Isolated from Cats

Cats are susceptible to a wide range of influenza A viruses (IAV). Furthermore, cats can serve as an intermediate host, and transfer avian influenza virus (AIV) H7N2 to a veterinarian. In this report, a novel reassortant influenza virus, designated A/feline/Jiangsu/HWT/2017 (H3N2), and abbreviated as FIV-HWT-2017, was isolated from nasal swab of a symptomatic cat in Jiangsu province, China. Sequence analysis indicated that, whilst the other seven genes were most similar to the avian-origin canine influenza viruses (CIV H3N2) isolated in China, the NS gene was more closely related to the circulating human influenza virus (H3N2) in the region. Therefore, FIV-HWT-2017 is a reassortant virus. In addition, some mutations were identified, and they were similar to a distinctive CIV H3N2 clade. Whether these cats were infected with the reassortant virus was unknown, however, this random isolation of a reassortant virus indicated that domestic or stray cats were “mixing vessel” for IAV cannot be ruled out. An enhanced surveillance for novel influenza virus should include pet and stray cats.

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

Host Adaptive Evolution of Avian-Origin H3N2 Canine Influenza Virus

Since its first isolation in around 2007, the avian-origin H3N2 canine influenza virus (CIV) has become established and continues to circulate in dog populations. This virus serves as a useful model for deciphering the complex evolutionary process of interspecies transmission of influenza A virus (IAV) from one species to its subsequent circulation in another mammalian host. The present investigation is a comprehensive effort to identify and characterize genetic changes that accumulated in the avian-origin H3N2 CIV during its circulation in the dog. We revealed that H3N2 CIV experiences greater selection pressure with extremely high global non-synonymous to synonymous substitution ratios per codon (dN/dS ratio) for each gene compared to the avian reservoir viruses. A total of 54 amino acid substitutions were observed to have accumulated and become fixed in the H3N2 CIV population based on our comprehensive codon-based frequency diagram analysis. Of these substitutions, 11 sites also display high prevalence in H3N8 CIV, indicating that convergent evolution has occurred on different lineages of CIV. Notably, six substitutions, including HA-G146S, M1-V15I, NS1-E227K, PA-C241Y, PB2-K251R, and PB2-G590S, have been reported to play imperative roles in facilitating the transmission and spillover of IAVs across species barriers. Most of these substitutions were found to have become fixed in around 2015, which might have been a favorable factor that facilitating the spread of these CIV lineages from South Asia to North America and subsequent further circulation in these areas. We also detected 12 sites in six viral genes with evidence for positive selection by comparing the rates of non-synonymous and synonymous substitutions at each site. Besides, our study reports trends of enhanced ongoing adaptation of H3N2 CIV to their respective host cellular systems, based on the codon adaptation index analysis, which points toward increasing fitness for efficient viral replication. In addition, a reduction in the abundance of the CpG motif, as evident from an analysis of relative dinucleotide abundance, may contribute to the successful evasion of host immune recognition. The present study provides key insights into the adaptive changes that have accumulated in the avian-origin H3N2 viral genomes during its establishment and circulation into dog populations.