Ann Cullinane

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.

The DNA sequence of equine herpesvirus-4.

The complete DNA sequence of equine herpesvirus-4 (EHV-4) strain NS80567 was determined. The genome is 145597 bp in size and consists of a long unique region (UL, 112398 bp) flanked by a short inverted repeat (TRL/IRL, 27 bp) linked to a short unique region (Us, 12789 bp) flanked by a substantial inverted repeat (TRs/IRs, 10178 bp). EHV-4 is predicted to contain 76 different genes; three of these are present twice in TRs/IRs, giving a total of 79 genes. The closely related virus equine herpesvirus-1 (EHV-1) also possesses 76 different genes corresponding to those of EHV-4, but has a total of 80 genes because four are present twice in TRs/IRs. Interpretations of the coding capacity of the EHV-4 and EHV-1 genomes were refined by comparing the complete DNA sequences.

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.

Equine influenza virus infections: An update

Summary Equine influenza is one of the most economically important contagious respiratory diseases of horses. In this paper the current state of knowledge of equine influenza virus and the most important aspects of these virus infections, e.g. epidemiology, clinical aspects, pathogenesis and pathology, immunity, diagnosis, treatment, management and vaccination, are reviewed with an emphasis on epidemiology, diagnosis and vaccinology. Many questions have remained and with the advent of improved technology new questions have arisen. Consequently, research priorities should be set in an attempt to answer them. Therefore, this review ends with some personal recommendations for important priorities for future research.

Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4). Vaccination/challenge experiments in foals and pregnant mares

Pregnant mares and young foals were vaccinated with Duvaxyn EHV1,4, an inactivated and adjuvanted vaccine containing both the EHV-1 and 4 antigens. SN and CF antibody titres were induced two weeks after first vaccination. Antibody levels were boosted after second vaccination, however they never reached the levels induced after virus challenge. Young foals were challenged with virulent EHV-1 and EHV-4 field viruses. Pregnant mares were challenged with the highly abortigenic EHV-1 strain Ab4. Vaccinated animals showed a clear reduction in clinical signs and virus excretion compared to unvaccinated control animals. Log transformed antibody levels could be correlated to duration of virus excretion. The incidence of EHV-1 induced abortions was drastically reduced in vaccinated mares. Therefore, although vaccinated animals are not fully protected against disease, Duvaxyn EHV1,4 clearly reduces clinical symptoms, the duration of virus shedding and the quantity of virus shed. It can be concluded that vaccination of foals and pregnant mares with Duvaxyn EHV1,4 significantly reduces the risk of abortions and outbreaks of respiratory disease caused by circulating field viruses.

Characterization of the genome of equine herpesvirus 1 subtype 2.

The genome structure of equine herpesvirus 1 (EHV-1) subtype 2 was shown by electron microscopic studies and restriction endonuclease site mapping to comprise two covalently linked segments (L, 109 kbp; S, 35 kbp). The S segment contains a unique sequence (US) flanked by a substantial inverted repeat (TRS/IRS). Thus, the genome structure of EHV-1 subtype 2 is similar to that published previously for EHV-1 subtype 1, but the two subtypes differ in the occurrences of EcoRI and BamHI restriction sites. Hybridization studies using cloned EHV-1 DNA showed that the genome of EHV-1 subtype 2 is colinear with the genomes of EHV-1 subtype 1 and herpes simplex virus type 1. DNA sequence data for four EHV-1 subtype 2 genes, including one potentially encoding a glycoprotein, were obtained by sequencing a 4574 bp BamHI fragment containing the junction between US and TRS. The genome structure, hybridization and sequence data confirm that EHV-1 subtype 2 is of the alphaherpesvirus lineage.

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.