Michelle Quinlivan

Attenuation of Equine Influenza Viruses through Truncations of the NS1 Protein

ABSTRACT Equine influenza is a common disease of the horse, causing significant morbidity worldwide. Here we describe the establishment of a plasmid-based reverse genetics system for equine influenza virus. Utilizing this system, we generated three mutant viruses encoding carboxy-terminally truncated NS1 proteins. We have previously shown that a recombinant human influenza virus lacking the NS1 gene (delNS1) could only replicate in interferon (IFN)-incompetent systems, suggesting that the NS1 protein is responsible for IFN antagonist activity. Contrary to previous findings with human influenza virus, we found that in the case of equine influenza virus, the length of the NS1 protein did not correlate with the level of attenuation of that virus. With equine influenza virus, the mutant virus with the shortest NS1 protein turned out to be the least attenuated. We speculate that the basis for attenuation of the equine NS1 mutant viruses generated is related to their level of NS1 protein expression. Our findings show that the recombinant mutant viruses are impaired in their ability to inhibit IFN production in vitro and they do not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in MDCK cells, or in vivo in a mouse model. Therefore, these attenuated mutant NS1 viruses may have potential as candidates for a live equine influenza vaccine.

Dynamics of Influenza Virus Infection and Pathology

ABSTRACT A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon (“cytokine storms”), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.

Modeling Within-Host Dynamics of Influenza Virus Infection Including Immune Responses

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection.

Comparison of Sensitivities of Virus Isolation, Antigen Detection, and Nucleic Acid Amplification for Detection of Equine Influenza Virus

ABSTRACT Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 106 50% egg infective doses (EID50)/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID50 was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucleoprotein gene and a single set of primers from the matrix gene. RT-PCR using the matrix primers and virus isolation in embryonated eggs proved to be the most sensitive methods for the detection of virus. The Directigen Flu A test was the least sensitive method. The inclusion of 2% fetal calf serum in the viral transport medium inhibited the growth of virus from undiluted samples in MDCK cells but was essential for the maintenance of the virus titer in samples subjected to repeated freeze-thaw cycles.

Real-time reverse transcription PCR for detection and quantitative analysis of equine influenza virus.

ABSTRACT Equine influenza is a cause of epizootic respiratory disease of the equine. The detection of equine influenza virus using real-time Light Cycler reverse transcription (RT)-PCR technology was evaluated over two influenza seasons with the analysis of 171 samples submitted for viral respiratory disease. Increased sensitivity was found in overall viral detection with this system compared to Directigen Flu A and virus isolation, which were 40% and 23%, respectively, that of the RT-PCR. The assay was also evaluated as a viable replacement for the more traditional methods of quantifying equine influenza virus, 50% egg infectious dose and 50% tissue culture infectious dose. There was a significant positive correlation (P < 0.05) between the quantitative RT-PCR and both of these assays.

Influenza A viruses with truncated NS1 as modified live virus vaccines: pilot studies of safety and efficacy in horses.

REASONS FOR PERFORMING STUDY Three previously described NS1 mutant equine influenza viruses encoding carboxy-terminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. HYPOTHESIS One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild-type influenza have reduced physiological and virological correlates of disease. METHODS Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. RESULTS Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1-73 and NS1-126 viruses, but not the NS1-99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild-type influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5 degrees C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL-1beta and IL-6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. CONCLUSION AND CLINICAL RELEVANCE These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics-based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.

Real-time quantitative rt-pcr and pcr assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene

In 2006, an outbreak of equine infectious anaemia (eia) occurred in Ireland. The initial source of the outbreak is believed to have been contaminated plasma imported from Italy. This paper presents the nucleotide sequence of the gag gene of the virus identified in Ireland (eiavire), the first for a European strain of eiav. Comparison of the gag gene with North American and Asian strains of the virus showed that the gag gene is less well conserved than previously believed, and that eiav strains can have similar phenotypes despite considerable variations in genotype. On the basis of the deduced sequence of the eiavire gag gene, highly sensitive, specific and quantitative rt-pcr and pcr assays were developed, and used to quantify the eiav nucleic acid in postmortem tissues, plasma and secretions of infected horses. This is the first report of the detection and quantification of eiav in nasal, buccal and genital swabs by rt-pcr.

The molecular epidemiology of equine influenza in Ireland from 2007–2010 and its international significance

REASONS FOR PERFORMING THE STUDY Antigenic and genetic drift of equine influenza (EI) virus is monitored annually by the Expert Surveillance Panel (ESP), which make recommendations on the need to update vaccines. Surveillance programmes are essential for this process to operate effectively and to decrease the risk of disease spread through the international movement of subclinically infected vaccinated horses. Not only is surveillance necessary to inform vaccine companies which strains are in circulation, but it serves as an early warning system for horse owners, trainers and veterinary clinicians, facilitating the implementation of appropriate prophylactic and control measures. OBJECTIVE To summarise the genetic analysis of EI viruses detected in Ireland from June 2007 to January 2010. METHODS The HA1 gene of 18 viruses was sequenced and phylogenetic analysis undertaken. RESULTS All viruses belonged to the Florida sublineage of the American lineage. Clade 2 viruses predominated up to 2009. The viruses identified on 4 premises in 2007 displayed 100% nucleotide identity to A/eq/Richmond/1/07, the current clade 2 prototype. The first clade 1 virus was identified in November 2009 and, thereafter, clade 1 viruses were responsible for all the outbreaks identified. The Irish clade 1 viruses differ from the clade 1 virus responsible for the EI outbreaks in Japan and Australia in 2007. No virus of the Eurasian lineage was isolated during this surveillance period. CONCLUSIONS In 2010 the ESP recommended that the vaccines should not include a H7N7 virus or a H3N8 virus of the Eurasian lineage but that they should contain both a clade 1 and clade 2 virus of the Florida sublineage. The surveillance data presented here support these recommendations and indicate that they are epidemiologically relevant. POTENTIAL RELEVANCE These data also serve as a scientific basis for investigating the source of epizootics and outbreaks both nationally and internationally.

Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland

In 2006 there was an outbreak of equine infectious anaemia (eia) in Ireland. This paper describes the use of the diagnosis of clinical and subclinical cases of the disease. In acute cases the elisas and the immunoblot were more sensitive than the agid. In one mare, fluctuating antibody levels were observed in all the serological assays before it seroconverted by agid. Viral rna and dna were detected by rt-pcr and pcr in all the tissues from the infected animals examined postmortem. The pcr detected viral dna in plasma regardless of the stage of the disease. In contrast, the rt-pcr detected rna in only 52 per cent of the seropositive animals tested and appeared to be most sensitive for the detection of virus early in infection. Both pcr and rt-pcr demonstrated potential to detect acutely infected horses earlier than some of the official tests. The serological data suggest that the usual incubation/seroconversion period for this strain of the virus was approximately 37 days but may be more than 60 days in a few cases.

Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland

Equine infectious anemia virus (EIAV), the causative agent of equine infectious anaemia (EIA), possesses the least-complex genomic organization of any known extant lentivirus. Despite this relative genetic simplicity, all of the complete genomic sequences published to date are derived from just two viruses, namely the North American EIAV(WYOMING) (EIAV(WY)) and Chinese EIAV(LIAONING) (EIAV(LIA)) strains. In 2006, an outbreak of EIA occurred in Ireland, apparently as a result of the importation of contaminated horse plasma from Italy and subsequent iatrogenic transmission to foals. This EIA outbreak was characterized by cases of severe, sometimes fatal, disease. To begin to understand the molecular mechanisms underlying this pathogenic phenotype, complete proviral genomic sequences in the form of 12 overlapping PCR-generated fragments were obtained from four of the EIAV-infected animals, including two of the index cases. Sequence analysis of multiple molecular clones produced from each fragment demonstrated the extent of diversity within individual viral genes and permitted construction of consensus whole-genome sequences for each of the four viral isolates. In addition, complete env gene sequences were obtained from 11 animals with differing clinical profiles, despite exposure to a common EIAV source. Although the overall genomic organization of the Irish EIAV isolates was typical of that seen in all other strains, the European viruses possessed ≤80 % nucleotide sequence identity with either EIAV(WY) or EIAV(LIA). Furthermore, phylogenetic analysis suggested that the Irish EIAV isolates developed independently of the North American and Chinese viruses and that they constitute a separate monophyletic group.