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This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

H5N1 highly pathogenic avian influenza (HPAI) virus causes high mortality of infected birds, with infection in multiple organs, including in feathers. Feathers have been proposed as samples for diagnosis of HPAI infection in birds, and this study is part of a broader investigation validating the use of feathers for diagnostic purposes. To understand and characterize the morphological basis for feather infection, sections from 7 different skin tracts of ducks and chickens infected with 3 different clades of H5N1 HPAI virus from Indonesia and Vietnam were examined histologically. Results showed that in ducks, lesions and viral antigen were mainly detected in the epidermis of feathers and follicles, whereas in chickens, they were mostly found in the dermis of these structures. Abundant viral antigen was found in nearly all the feathers examined from chickens, and there was no apparent difference between virus isolates or skin tracts in the proportion of feathers that were antigen positive. By immunohistochemistry, the majority of feathers from most skin tracts from ducks infected with a Vietnamese H5N1 HPAI virus contained abundant levels of viral antigen, while few feathers were antigen positive from ducks infected with 2 Indonesian viruses. These results support and inform the use of feathers for diagnostic detection of H5N1 HPAI virus in birds.

Aim: Current therapies against avian influenza (H5N1) provide limited clinical benefit. FBF-001 is a highly purified equine polyclonal immunoglobulin fragment against H5N1. Methods: Using a ferret model of severe acute H5N1 infection, we assessed FBF-001 when administered on the same day or 1 day after viral challenge, in comparison with oseltamivir therapy. Results: Untreated animals died 2–3 days after challenge. FBF-001 prevented most severe illness and reduced nasal viral load, with best efficacy when administered on the day of viral challenge. Oseltamivir and FBF-001 had synergistic impact on survival. Conclusion: FBF-001 prevented severe consequences of lethal H5N1 challenge in ferrets by controlling viral replication, an effect synergistic to oseltamivir. FBF-001 has recently been granted EMA orphan drug status.

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease. IMPORTANCE Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

Human infections with highly pathogenic avian influenza (HPAI) H5N1 have been associated with central nervous system involvement. The purpose of this study was to examine the route of invasion of wild-type HPAI H5N1 virus into the central nervous system (CNS) using a ferret model of infection. Sixteen ferrets were exposed by the intranasal route to 10(6) TCID(50) of A/Vietnam/1203/04, a Clade 1 strain originally isolated from a fatal human case. The ferrets were euthanased for histological and virological analysis at intervals after challenge at 1, 3, 5, 6 and 7 days post-inoculation (dpi). From 5 dpi encephalitis was seen in all examined ferrets. The detection of antigen in the olfactory epithelium, the olfactory bulb, and related nuclei, in that temporal sequence, supported the contention that this is a major infection route for this virus strain. The detection of antigen in the epithelial cells in the Eustachian tube on 1 dpi, followed by the cochlea and vestibulocochlear nerve on 5 dpi is consistent with a second anterograde route of invasion, namely the vestibulocochlear pathway. There was also antigen in the lining of the ventricles and central canal indicating spread via the cerebrospinal fluid. However, evidence for haematogenous dissemination in the form of antigen in the brain parenchyma surrounding blood vessels was not found. This study provides support to the contention that wild-type HPAI H5N1 virus strains may enter the CNS via cranial nerve pathways and that the ferret is an appropriate model to study preventive and therapeutic procedures involving neural infection with these viruses by this route.