Host Immune Response Against EHV-1: A Novel Approach

Hongkong plans The Research Problem: Equid alphaherpesvirus 1 (EHV-1) is a viral respiratory tract disease of equines of all ages. EHV-1 spreads horizontally through direct smear infection, fomite transmission, as well as via droplets and aerosols. Least is known about the latter. Secondary complications can follow primary infection include spinal cord disease (Equine herpesvirus-associated myeloencephalopathy, EHM) leading to paralysis and euthanasia, or, in a milder form, disease that may affect (permanently) the horse’s gait. Another important complication of EHV-1 infection is late-stage abortion or neonatal death. These complications are linked to systemic viral spread, viremia. Because of significant emotional and economic impact of all aspects of the equine industry quarantine measures for 4 to 6 weeks with travel restrictions are recommended, while outbreaks at racecourses or equestrian events lead to cancelations and on-site quarantine of affected and in-contact horses. EHV-1 infections in a herd can be clinical or subclinical. When clinical, the spectrum of disease varies from mild rhinitis to high fevers and systemic vasculitis for several days, followed in some by EHM (or abortion if pregnant mares are on site). While over the years our group and others have identified risk factors for the development of EHM (age, sex, breed, immune profile), an important factor for the diverse presentation of clinical signs also likely includes the infectious dose. At the end of quarantine of an outbreak it has been noticed that ≥90% of horses sharing the same airspace have seroconverted (van Maanen 2001), while only a small proportion, 20 -30% showed clinical signs of systemic disease. An even smaller proportion of ≤10% showed neurological signs. Our group has been involved/consulted with numerous EHV-1 outbreaks with EHM. is the large proportion of EHM cases during the first half of an outbreak when compared to the second half. In addition, the most severe cases of EHM, recumbency and tetraparalysis, are detected at the very beginning of an outbreak followed by milder presentations with ataxia only following the index case (Goehring 2006, Couroucé 2022). We believe that variation in infectious dose is responsible for this effect. An infectious dose (ID) is the number of infectious particles delivered, and a total dose would be better depicted as an ‘infectious dose -area under the curve’ (iAUC). EHV-1 infectious particles, virions, are the result of viral replication and assembly within the epithelium of the respiratory tract. At threshold an epithelial cell ruptures and releases new virions into the respiratory tract mixing with mucus and secretions. EHV-1 is expelled from the respiratory tract mixed with nasal secretions with most being deposited as smear in the direct surroundings of the horse. However, virion is also likely to leave the respiratory tract as droplets and aerosols. Cov-19 research has shown that the amount of virus that leaves the human respiratory tract during this time period, will constantly change in its ratio of droplets-to-aerosol particles depending on respiratory rate, depth and activity (e.g. regular resting breathing, vocalization, coughing, sneezing, but also cool down after exercising activities). Droplets differ from aerosols in size and in their viral load. Aerosols can be between few and 100um in diameter. Covid-19 research has further revealed that droplets, due to their weight, stay close to the shedding source, however, aerosols travel further and stay longer within a closed airspace (see a computer simulation by Aalto University, Finland). Although aerosol particles seem to play a less important role in effective transmission, in EHV-1 transmission they still could have an important role in immune system priming which could be an explanation for fewer or less severe EHM cases during the second half of an outbreak. There are 2 further aspects of aerosols why they should be investigated in the context of EHV-1 transmission and clinical disease. First, upon collision with other droplet/aerosol particles they can again increase in size, and with (indoor) temperature drops at night reaching dewpoint conditions, there will be condensation. We currently don’t know the tributary role of condensate as a source of infection spread. Secondly, small aerosol particles, when inhaled, are able to reach the most distant parts of the respiratory tract: the terminal bronchioles and alveoli. Studies in swine have shown that antigen presentation is different between the upper and lower respiratory tract. Currently, experimental (challenge) infections are induced via nasopharyngeal instillation with a high virus titer. Alternatives are the use of an inhalation chamber where horses are exposed (for ≤1 hr) to an infectious mist. Here, infection is localized to the upper respiratory tract. Two studies have used an inhalation model using face masks connected to a jet nebulizer creating a mix of droplets and aerosol. Both studies used adult horses for infection where it is uncommon to see primary fevers (Hussey ‘old vs young’). On the contrary, both studies reported high primary fevers, while in the study by Goehring et al. (2010) there was only one out of 4 horses that also developed a secondary fever, viremia, and subsequently EHM. We currently don’t know the impact of different (in quality and quantity) infectious doses have on immune response and clinical outcome. We have limited comprehension on shared airspace and indirect transmission. For distance between horses, we currently rely on ‘more is better’ and the removal of a shedding horse from shared airspace. However, ‘space’ can be very limited, and we need to find ways of effective intervention. This project has global impact on equine husbandry, whether it is at boarding facilities, equestrian events race courses, and veterinary care facilities, including impact for horse transportation. The project also will not only broaden our understanding of the relationship between dose, clinical signs and immunity/immunopathology, but will also have impact on prevention and mitigation strategies. The overarching research question we are asking is: How does ‘EHV-1 infectious dose’ and mode of infection affect clinical disease and the host immune response. We hypothesize that: Differences in duration of exposure, deposition and dose of EHV-1 will result in differences in clinical outcome and immune responses. We hypothesize that differences in the infectious dose quantity and quality, including a time factor of exposure, will result in different host immune system responses and clinical outcome. With this proposal we formulated 3 specific aims: Specific aim 1: we will determine if a low infectious dose is more likely to result and will affect magnitude and duration of viremia. Specific aim 2: we will determine how deposition of an infectious inoculum at different locations of the horse’s respiratory tract (upper vs. lower respiratory tract) affects clinical outcome and immune response. Specific aim 3: We will study how airborne transmission (droplets and aerosols) cause infection in sentinel horses exposed to a shedder. We will determine how differs between droplets and aerosol exposure. and condensate in a single infectious airspace. To accomplish these aims, we will use EHV-1 naïve yearling horses in all specific aims that are seronegative for EHV-1 (-4). They will be infected with EHV-1 strain Ab4 in 4 separate experiments. Measurable outcomes will be the comparison of pre and post exposure for clinical parameters including fever, respiratory disease and secondary manifestations. In addition, we will determine quantity and duration of nasal virus shedding (PCR), viremia (PCR), SAA, cytokines/chemokines in nasal fluids and PBMC, seroconversion, IgG 4/7 vs 3/5. We will also collect environmental samples (including condensate), and we will compare EHV-1 genome presence with ‘infectivity’ via viral isolation and quantitation. All this information is crucial to re-define experimental infections and vaccination-challenge experiments, as we may have looked at overwhelming and unrealistic infectious dose. Furthermore, investigating into indirect airborne transmission we will be able to identify ways and test towards an effective mitigation in the future.