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.

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.

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.

These data also serve as a scientific basis for investigating the source of epizootics and outbreaks both nationally and internationally.

SummaryReasons for performing study-A reverse genetics rescue system for equine influenza virus and the construction of three NS1 mutant viruses encoding carboxy-terminally truncated NS1 proteins of 73, 99 or 126 amino acids, have been previously described (Quinlivan et al., J. Virology 79, 8431-8439, 2005). These viruses are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, thus are candidates for use as a modified live influenza virus vaccine in the horse.Hypothesis-One or more of these mutant viruses are 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 pro-inflammatory 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 wildtype influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5°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. Expression of proinflammatory cytokines IL-1β, IL-6, IFNγ, and TNFα was examined by quantitative RT-PCR of mRNA. Mean IL-1β and IL-6 levels were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post-challenge.Conclusion-These data suggest the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza.Relevance-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.

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.

Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/ Kildare/89 at 10 6 50% egg infective doses (EID 50 )/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID 50 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.Equine influenza is considered to be the most important respiratory disease of the horse in the majority of countries where the breeding and racing of horses is a major industry. There are two subtypes of equine influenza virus, which is an orthomyxovirus: A/Equi 1/H7N7, first isolated in 1956 (20), and A/Equi 2/H3N8, first isolated in 1963 (24). Both subtypes have caused disease. However, it is generally accepted that A/Equi 1/H7N7 has not been isolated since 1979 and may be extinct (22,26). In contrast, A/Equi 2/H3N8 continues to circulate worldwide with the exception of a small number of island countries, such as Australia, New Zealand, and Iceland, where equine influenza has never been recorded. Infection with this subtype appears to be enzootic in North America and Europe, where two separate virus lineages have evolved and outbreaks frequently occur despite the mandatory vaccination of some horse populations (2, 10, 16a). However, it is immunologically naïve populations that are most at risk from equine influenza. Influenza is highly contagious, and the introduction of a single infected horse can result in explosive virus spread in unprotected horses over a wide geographical area. In South Africa in 1986, the introduction of the virus into the country for the first time resulted in thousands of horses suffering severe respiratory disease and necessitated the cancellation of racing for 5 months (5, 18).The risk associated with the increase in the international movement of horses by air transport for racing and breeding purposes means that it is imperative that there are sensitive virus detection systems available for the rapid diagnosis of equine influenza (21). Traditionally, equine influenza virus is diagnosed by the isolation of virus from nasopharyngeal swabs in embryonated hen eggs or by the detection of a fourfold-orgreater rise in antibody titer in paired sera by hemagglutination inhibition (16).The main objective of ...

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.