Gabriele A. Landolt

Comparison of the Pathogenesis of Two Genetically Different H3N2 Influenza A Viruses in Pigs

ABSTRACT In 1997 and 1998, H3N2 influenza A viruses emerged among pigs in North America. Genetic analyses of the H3N2 isolates demonstrated that they had distinctly different genotypes. The most commonly isolated viruses in the United States have a triple-reassortant genotype, with the hemagglutinin, neuraminidase, and PB1 polymerase genes being of human influenza virus origin, the nucleoprotein, matrix, and nonstructural genes being of classical swine influenza virus origin, and the PA and PB2 polymerase genes being of avian influenza virus origin. In contrast, a wholly human H3N2 virus was isolated from a single baby pig in Ontario, Canada, in 1997, but it did not spread within the swine population. Genetic differences between this wholly human virus and the triple-reassortant viruses may affect their replication efficiencies in pigs. In the present study we compared the pathogenicities and replication kinetics of the wholly human virus and a triple-reassortant virus in 7-week-old pigs that were infected intranasally with 2 × 103 to 2 × 106 50% tissue culture infective doses of virus. Our results demonstrate that the wholly human virus replicated to significantly lower titers and that the onset of virus shedding was delayed compared to the replication titers and the time of onset of virus shedding in triple-reassortant viruses. In addition, infection with the triple-reassortant virus was associated with moderate to severe gross pathological and histological pulmonary lesions, while infection with the wholly human virus induced only mild pulmonary changes.

Up to new tricks – A review of cross-species transmission of influenza A viruses

Abstract Influenza is a highly contagious disease that has burdened both humans and animals since ancient times. In humans, the most dramatic consequences of influenza are associated with periodically occurring pandemics. Pandemics require the emergence of an antigenically novel virus to which the majority of the population lacks protective immunity. Historically, influenza A viruses from animals have contributed to the generation of human pandemic viruses and they may do so again in the future. It is, therefore, critical to understand the epidemiological and molecular mechanisms that allow influenza A viruses to cross species barriers. This review summarizes the current knowledge of influenza ecology, and the viral factors that are thought to determine influenza A virus species specificity.

Detection and Management of an Outbreak of Equine Herpesvirus Type 1 Infection and Associated Neurological Disease in a Veterinary Teaching Hospital

BACKGROUND Because of the serious disease sequelae associated with equine herpesvirus type 1 (EHV-1) infections, awareness and control measures used to control outbreaks are important issues for all horse populations. OBJECTIVES Describe the occurrence and management of an outbreak of EHV-1 infection at a veterinary hospital. ANIMALS Horses hospitalized at a referral veterinary hospital. METHODS A horse with myeloencephalopathy associated with EHV-1 infection (EHM) was admitted for diagnostic evaluation and treatment under strict infection control procedures. We describe the occurrence and management of a nosocomial outbreak of EHV-1 infections associated with admission of this patient. RESULTS Despite institution of rigorous biosecurity precautions at the time of admission of the index case, EHV-1 infections spread to 6 other horses that were hospitalized at the James L. Voss Veterinary Teaching Hopsital, including 2 that served as sources of infection for horses on their home premises after discharge. Infection with EHV-1 was confirmed by polymerase chain reaction (PCR) and by seroconversion documented by glycoprotein G ELISA. A voluntary quarantine was imposed and admissions were restricted to prevent additional horses from being exposed. Quarantine duration was abbreviated by serial testing of all horses with PCR. CONCLUSIONS AND CLINICAL IMPORTANCE These findings illustrate the contagious disease risk that can accompany management of horses with EHM. Horses with active nasal EHV-1 shedding should be isolated in an airspace that is separate from other horses by strictly enforced biosecurity and isolation procedures. Serial testing with PCR may be a useful adjunct to determine when the risk of transmission has been minimized.

Onset and duration of immunity to equine influenza virus resulting from canarypox-vectored (ALVAC) vaccination.

Equine influenza virus remains an important problem in horses despite extensive use of vaccination. Efficacy of equine influenza vaccination depends on the onset and duration of protective immunity, and appropriate strain specificity of the immune response. This study was designed to test the protective immunity resulting from vaccination with the North American commercial ALVAC equine influenza vaccine (RECOMBITEK Influenza, Merial, USA)(1) against challenge with American lineage influenza viruses. In experiment 1, 12 ponies were vaccinated twice, at a 35 day interval, using the ALVAC-influenza vaccine expressing the HA genes of influenza A/eq/Newmarket/2/93 and A/eq/Kentucky/94 (H3N8), and 11 ponies served as unvaccinated controls. Six months after the second vaccination, all ponies were challenged with A/eq/Kentucky/91. In experiment 2, 10 ponies received one dose of the ALVAC-influenza vaccine, 10 ponies served as unvaccinated controls, and all ponies were challenge infected with A/equine/Ohio/03, 14 days after vaccination. Parameters studied included serological responses, and clinical disease and nasal viral shedding following challenge infection. In experiment 1, following the two-dose regimen, vaccinated ponies generated high titered anti-influenza virus IgGa and IgGb antibody responses to vaccination and demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. Infection with A/eq/Kentucky/91 produced unusually severe signs in ponies in the control group, requiring therapy with NSAIDs and antibiotics, and leading to the euthanasia of one pony. In experiment 2 following the one-dose regimen, vaccinates generated IgGa responses pre-challenge, and anamnestic IgGa and IgGb responses after challenge. Vaccinates demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. The results of this study clearly demonstrate the early onset, and 6-month duration of protective immunity resulting from ALVAC-influenza vaccination against challenge with American lineage equine influenza viruses.

Restricted Infectivity of a Human-Lineage H3N2 Influenza A Virus in Pigs Is Hemagglutinin and Neuraminidase Gene Dependent

ABSTRACT Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses.

Evaluation of infectivity of a canine lineage H3N8 influenza A virus in ponies and in primary equine respiratory epithelial cells

OBJECTIVE To evaluate whether an equine-derived canine H3N8 influenza A virus was capable of infecting and transmitting disease to ponies. ANIMALS 20 influenza virus-seronegative 12- to 24-month-old ponies. PROCEDURES 5 ponies were inoculated via aerosol exposure with 10(7) TCID(50) of A/Canine/Wyoming/86033/07 virus (Ca/WY)/pony. A second group of 5 ponies (positive control group) was inoculated via aerosol exposure with a contemporary A/Eq/Colorado/10/07 virus (Eq/CO), and 4 sham-inoculated ponies served as a negative control group. To evaluate the potential for virus transmission, ponies (3/inoculation group) were introduced 2 days after aerosol exposure and housed with Ca/WY- and Eq/CO-inoculated ponies to serve as sentinel animals. Clinical signs, nasal virus shedding, and serologic responses to inoculation were monitored in all ponies for up to 21 days after viral inoculation. Growth and infection characteristics of viruses were examined by use of Madin-Darby canine kidney cells and primary equine and canine respiratory epithelial cells. RESULTS Ponies inoculated with Ca/WY had mild changes in clinical appearance, compared with results for Eq/CO-inoculated ponies. Additionally, Ca/WY inoculation induced significantly lower numbers for copies of the matrix gene in nasal secretions and lower systemic antibody responses in ponies than did Eq/CO inoculation. The Ca/WY isolate was not transmitted to sentinel ponies. CONCLUSIONS AND CLINICAL RELEVANCE Inoculation of ponies with the canine H3N8 isolate resulted in mild clinical disease, minimal nasal virus shedding, and weak systemic antibody responses, compared with responses after inoculation with the equine H3N8 influenza isolate. These results suggested that Ca/WY has not maintained infectivity for ponies.

In Vitro Susceptibility of Canine Influenza A (H3N8) Virus to Nitazoxanide and Tizoxanide

Infection of dogs with canine influenza virus (CIV) is considered widespread throughout the United States following the first isolation of CIV in 2004. While vaccination against influenza A infection is a common and important practice for disease control, antiviral therapy can serve as a valuable adjunct in controlling the impact of the disease. In this study, we examined the antiviral activity of nitazoxanide (NTZ) and tizoxanide (TIZ) against three CIV isolates in vitro. NTZ and TIZ inhibited virus replication of all CIVs with 50% and 90% inhibitory concentrations ranging from 0.17 to 0.21 μM and from 0.60 to 0.76 μM, respectively. These results suggest that NTZ and TIZ are effective against CIV and may be useful for treatment of canine influenza in dogs but further investigation of the in vivo efficacy against CIV as well as the drugs potential for toxicity in dogs is needed.

Epidemiology and Ecology of H3N8 Canine Influenza Viruses in US Shelter Dogs

Background H3N8 canine influenza virus (CIV) infection might contribute to increased duration of shelter stay for dogs. Greater understanding of factors contributing to CIV within shelters could help veterinarians identify control measures for CIV. Objectives To assess community to shelter dog CIV transmission, estimate true prevalence of CIV, and determine risk factors associated with CIV in humane shelters. Animals 5,160 dogs upon intake or discharge from 6 US humane shelters, December 2009 through January 2012. Methods A cross‐sectional study was performed with prospective convenience sampling of 40 dogs from each shelter monthly. Nasal swabs and serum samples were collected. Hemagglutination inhibition and real‐time reverse transcriptase‐polymerase chain reaction assays were performed for each nasal and serum sample. True prevalence was estimated by stochastic latent class analysis. Logistic regression was used to identify risk factors associated with CIV shedding and seropositivity. Results Nasal swabs were positive from 4.4% of New York (NY), 4.7% of Colorado (CO), 3.2% of South Carolina, 1.2% of Florida, and 0% of California and Texas shelter dogs sampled. Seropositivity was the highest in the CO shelter dogs at 10%, and NY at 8.5%. Other shelters had 0% seropositivity. Information‐theoretic analyses suggested that CIV shedding was associated with region, month, and year (model weight = 0.95) and comingling/cohousing (model weight = 0.92). Conclusions and Clinical Importance Community dogs are a likely source of CIV introduction into humane shelters and once CIV has become established, dog‐to‐dog transmission maintains the virus within a shelter.

Innate immune responses of airway epithelial cells to infection with Equine herpesvirus-1

Equine herpesvirus-1 (EHV-1) is the cause of respiratory disease, abortion and myelitis in horses worldwide. Protection following infection or vaccination is typically incomplete and this lack of protective immunity is thought to be due to the immunomodulatory properties of EHV-1. EHV-1 immune modulation is likely initiated early in the infection cycle at the respiratory epithelium, but to date, immunity to EHV-1 at the epithelial cell barrier remains poorly characterized. Thus, the purpose of this study was to use a recently established primary equine respiratory epithelial cell culture (EREC) system to characterize innate immunity to EHV-1. Differentiated ERECs were inoculated with a neuropathogenic strain of EHV-1 and cytokine responses were determined using quantitative real-time polymerase chain reaction and ELISA. Major histocompatibility complex (MHC)-I and MHC-II as well as toll-like receptor (TLR)3 and TLR9 protein expression were examined using fluorescence activated cell-sorting analysis and chemotaxis of neutrophils and monocytes were evaluated using chemotaxis assays. Infection with EHV-1 resulted in increased expression of TLR3 and 9 as well as inflammatory cytokines (IL-1, TNF-alpha, IFN-alpha, and IL-6) and chemokines (IL-8, MCP-1). In contrast, EHV-1 infection caused marked decreases of MHC-I and MHC-II expression as well as a reduction in IFN-gamma production. In summary, these results provide an initial characterization of the early immune response to EHV-1 at the epithelial cell barrier and show that, while EHV-1 maintains induction of an inflammatory response, it causes an attenuation of IFN-gamma responses and down-modulates expression of MHC-I and MHC-II, which are important molecules for antigen presentation.

Comparison of the Infectivity and Transmission of Contemporary Canine and Equine H3N8 Influenza Viruses in Dogs

Phylogenetic analyses indicate that canine influenza viruses (CIVs) (H3N8) evolved from contemporary equine influenza virus (EIV). Despite the genetic relatedness of EIV and CIV, recent evidence suggests that CIV is unable to infect, replicate, and spread among susceptible horses. To determine whether equine H3N8 viruses have equally lost the ability to infect, cause disease, and spread among dogs, we evaluated the infectivity and transmissibility of a recent Florida sublineage EIV isolate in dogs. Clinical signs, nasal virus shedding, and serological responses were monitored in dogs for 21 days after inoculation. Real-time reverse transcription-PCR and hemagglutination inhibition assays showed that both the viruses have maintained the ability to infect and replicate in dogs and result in seroconversion. Transmission of EIV from infected to sentinel dogs, however, was restricted. Furthermore, both CIV and EIV exhibited similar sialic acid-α2,3-gal receptor-binding preferences upon solid-phase binding assays. The results of the in vivo experiments reported here suggesting that dogs are susceptible to EIV and previous reports by members of our laboratory showing limited CIV infection in horses have been mirrored in CIV and EIV infections studies in primary canine and equine respiratory epithelial cells.