Judy A. Mitchell

Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins

Cross-protection between different subtypes of influenza A virus has been attributed to heterosubtypic immunity (HSI). Although, HSI can occur in the absence of anti-HA or anti-NA antibodies, HSI seems to be mediated, in part, by cross-reactive antibodies. In this study, we examined the effects of a DNA vaccine expressing an influenza HA fused to three copies of murine C3d of complement (HA-mC3d(3)). HA-mC3d(3) elicited heterosubtypic immunity more efficiently than non-fused forms of HA and protected mice from lethal challenge of influenza with different subtypes. Plasmid encoding for various forms of HA were constructed from two influenza strains, A/Puerto Rico/8/34 (H1N1) or A/Aichi/2/68-x31 (H3N2). Vaccinated mice were analyzed for enhancement of anti-HA titers, affinity maturation of antibody, hemagglutinin-inhibition activity, and altered cytokine profiles. The HA-mC3d(3)-DNA vaccinated mice were protected from heterologous influenza challenge, even though sera from these mice had no viral-neutralizing activity against heterologous virus.

New and Emerging Pathogens in Canine Infectious Respiratory Disease

Canine infectious respiratory disease is a common, worldwide disease syndrome of multifactorial etiology. This review presents a summary of 6 viruses (canine respiratory coronavirus, canine pneumovirus, canine influenza virus, pantropic canine coronavirus, canine bocavirus, and canine hepacivirus) and 2 bacteria (Streptococcus zooepidemicus and Mycoplasma cynos) that have been associated with respiratory disease in dogs. For some pathogens a causal role is clear, whereas for others, ongoing research aims to uncover their pathogenesis and contribution to this complex syndrome. Etiology, clinical disease, pathogenesis, and epidemiology are described for each pathogen, with an emphasis on recent discoveries or novel findings.

Serological evidence for multiple strains of canine norovirus in the UK dog population.

Noroviruses are associated with intestinal disease in humans, cows, pigs, mice, and, more recently, dogs. In 2007, the first canine norovirus (CNV) was identified and characterized in Italy. Subsequent studies have identified CNV in stools of dogs from Portugal, Greece, and the United States. To investigate the prevalence of CNV in the UK dog population, 228 canine stool samples were screened for CNV by qPCR, and 396 serum samples were screened for anti-CNV antibodies. qPCR of RNA extracted from canine stool samples did not reveal any CNV-positive samples, based on samples collected from diarrhoeic and control dogs in 2012–2013. CNV virus-like particles to three different CNV strains were produced using recombinant baculoviruses and a seroprevalence screen undertaken. Anti-CNV antibodies were identified at significant levels in canine serum; 38.1% of samples collected between 1999–2001 and 60.1% of samples collected in 2012–2013 were seropositive. The increase in seroprevalence over time (p<0.001) suggests that the CNV strains screened for are becoming more widespread. Variation in seroprevalence to different CNV strains was also identified. Two-thirds of the dogs were seropositive to a single strain, whereas the remaining third were seropositive to two or three of the strains analysed. This study has provided the first evidence that CNV is present in the UK, with seroprevalence identified to multiple circulating strains. This warrants further study and increased awareness of this recently discovered canine virus.

Evidence for Human Norovirus Infection of Dogs in the United Kingdom

ABSTRACT Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244–247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.

Detection of non-primate hepaciviruses in UK dogs.

Abstract Non-primate hepacivirus (NPHV) has been identified in dogs, horses, bats and wild rodents. The presence of NPHV in dogs outside of the USA however is yet to be established. Here we describe for the first time the detection of NPHV in the UK dog population (described throughout the manuscript as CnNPHV). We examined tissues collected from dogs housed in a rehoming kennel where respiratory disease was endemic. CnNPHV RNA was detected in the tracheal tissues of 48/210 dogs by RT-PCR, and in the liver, lung and/or tracheal tissues of 12/20 dogs. The presence of CnNPHV RNA, and its tropism was confirmed by in situ hybridisation. Histopathological examination demonstrated a trend toward higher histopathological scores in CnNPHV RNA positive respiratory tissues, although, this was not statistically significant. Our findings broaden the geographic distribution and our understanding of CnNPHV. Further evidence of CnNPHV replication in canids warrants investigation.

Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV)

Abstract Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Kochs postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.

Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV)

Abstract One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-α and IL-6 and the chemokine IL-8 during the 72h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.

Development of a quantitative real-time PCR for the detection of canine respiratory coronavirus

Abstract Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV.

Detection of Canine Pneumovirus in Dogs with Canine Infectious Respiratory Disease

ABSTRACT Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n = 56/215 dogs) on the day of entry to 93.5% (n = 201/215 dogs) after 21 days (P <0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.

The challenges in developing effective canine infectious respiratory disease vaccines

Canine infectious respiratory disease (CIRD) is a disease of multifactorial aetiology, where multiple pathogens act sequentially or synergistically to cause disease. It is common within large dog populations, such as those in re‐homing or training kennels. Vaccines are vital in its management of CIRD, but they often fail to prevent disease. Recently, a number of novel pathogens have been identified in CIRD outbreaks and represent new targets for vaccination.