A Canine Distemper Outbreak in Urban Red Foxes (Vulpes vulpes), in Brussels, Belgium, 2020.

Canine distemper has been observed infrequently in Belgian wildlife, mainly stone martens (Martes foina) and red foxes (Vulpes vulpes). This report describes an outbreak in the Brussels urban red fox population, characterized by its high density. The identified virus matched those within a cluster of viruses found previously in red foxes in Germany. Different canine distemper virus (CDV) strains, found in Belgian wild carnivores, share relationships with viruses found farther east. This and other reports indicate an endemic distribution of CDV in wild carnivores in Europe whereby the complex interplay of population density, group immunity, and infection of metapopulations determines the pattern of spatiotemporally alternating outbreaks.

Infection with Pythium flevoense in a harbour porpoise (Phocoena phocoena) as a novel cause of dermatitis in marine mammals.

The oomycete Pythium flevoense was diagnosed as the cause of dermatitis in a young adult female harbour porpoise (Phocoena phocoena) that had been trapped in a pound net in a temperate saltwater environment. Disease from Pythium sp. infection-pythiosis-is infrequently diagnosed in humans, horses, dogs, cattle, and few other mammalian species. Pythiosis is typically associated with exposure to tropical or subtropical freshwater conditions, and typically caused by Pythium insidiosum. However, until now, pythiosis has been reported in neither marine mammals nor temperate saltwater conditions, and P. flevoense is not known as a cause of pythiosis in mammals. This porpoise developed generalised dermatitis despite treatment and euthanasia was necessary. Histopathological evaluation revealed a chronic active erosive dermatitis, with intralesional hyphae morphologically consistent with a Pythium sp. PCR analysis and sequencing of affected skin matched Pythium flevoense with a 100% similarity to the reference strain. Additional diagnostics excluded other pathogens. Based on this case report, P. flevoense needs to be considered as a mammalian pathogen. Furthermore, harbour porpoises and possibly other marine mammals may be at risk of infection with P. flevoense, and pythiosis should be included in the differential diagnosis of dermatitis in marine mammals.

Intestinal Tropism of a Betacoronavirus (Merbecovirus) in Nathusius's Pipistrelle Bat (Pipistrellus nathusii), Its Natural Host.

The emergence of several bat coronavirus-related disease outbreaks in human and domestic animals has fueled surveillance of coronaviruses in bats worldwide. However, little is known about how these viruses interact with their natural hosts. We demonstrate a Betacoronavirus (subgenus Merbecovirus), PN-ßCoV, in the intestine of its natural host, Nathusius's Pipistrelle Bat (Pipistrellus nathusii), by combining molecular and microscopy techniques. Eighty-eight P. nathusii bat carcasses were tested for PN-ßCoV RNA by RT-qPCR, of which 25 bats (28%) tested positive. PN-ßCoV RNA was more often detected in samples of the intestinal tract than in other sample types. In addition, viral RNA loads were higher in intestinal samples compared to other sample types, both on average and in each individual bat. In one bat, we demonstrated Merbecovirus antigen and PN-ßCoV RNA expression in intestinal epithelium and the underlying connective tissue using immunohistochemistry and in situ hybridization, respectively. These results indicate that PN-ßCoV has a tropism for the intestinal epithelium of its natural host, Nathusius's Pipistrelle Bat, and imply that the fecal-oral route is a possible route of transmission. IMPORTANCE Virtually all mammal species circulate coronaviruses. Most of these viruses will infect one host species; however, coronaviruses are known to include species that can infect multiple hosts, for example the well-known virus that caused a pandemic, SARS-CoV-2. Chiroptera (bats) include over 1,400 different species, which are expected to harbor a great variety of coronaviruses. However, we know very little about how any of these coronaviruses interact with their bat hosts; for example, we do not know their modes of transmissions, or which cells they infect. Thus, we have a limited understanding of coronavirus infections in this important host group. The significance of our study is that we learned that a bat coronavirus that occurs in a common bat species in Europe has a tropism for the intestines. This implies the fecal-oral route is a likely transmission route.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Long-Term Protective Effect of Serial Infections with H5N8 Highly Pathogenic Avian Influenza Virus in Wild Ducks.

Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong (Gs/Gd) lineage are an emerging threat to wild birds. In the 2016-2017 H5N8 outbreak, unexplained variability was observed in susceptible species, with some reports of infected birds dying in high numbers and other reports of apparently subclinical infections. This experimental study was devised to test the hypothesis that previous infection with a less-virulent HPAIV (i.e., 2014 H5N8) provides long-term immunity against subsequent infection with a more-virulent HPAIV (i.e., 2016 H5N8). Therefore, two species of wild ducks-the more-susceptible tufted duck (Aythya fuligula) and the more-resistant mallard (Anas platyrhynchos)-were serially inoculated, first with 2014 H5N8 and after 9 months with 2016 H5N8. For both species, a control group of birds was first sham inoculated and after 9 months inoculated with 2016 H5N8. Subsequent infection with the more-virulent 2016 H5N8 caused no clinical signs in tufted ducks that had previously been infected with 2014 H5N8 (n = 6) but caused one death in tufted ducks that had been sham inoculated (n = 7). In mallards, 2016 H5N8 infection caused significant body weight loss in previously sham-inoculated birds (n = 8) but not in previously infected birds (n = 7). IMPORTANCE This study showed that ducks infected with a less-virulent HPAIV developed immunity that was protective against a subsequent infection with a more-virulent HPAIV 9 months later. Following 2014 H5N8 infection, the proportion of birds with detectable influenza nucleoprotein antibody declined from 100% (8/8) in tufted ducks and 78% (7/9) in mallards after 1 month to 33% (2/6) in tufted ducks and 29% (2/7) in mallards after 9 months. This finding helps predict the expected impact that an HPAIV outbreak may have on wild bird populations, depending on whether they are immunologically naive or have survived previous infection with HPAIV.

Tropism of Highly Pathogenic Avian Influenza H5 Viruses from the 2020/2021 Epizootic in Wild Ducks and Geese.

Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species. The 2020/2021 epizootic was the largest and most deadly ever reported in Europe, and many new bird species tested positive for HPAI virus for the first time. This study investigated the tropism of HPAI virus in wild birds. We tested the pattern of virus attachment of 2020 H5N8 virus to intestinal and respiratory tissues of key bird species; and characterized pathology of naturally infected Eurasian wigeons (Mareca penelope) and barnacle geese (Branta leucopsis). This study determined that 2020 H5N8 virus had a high level of attachment to the intestinal epithelium (enterotropism) of dabbling ducks and geese and retained attachment to airway epithelium (respirotropism). Natural HPAI 2020 H5 virus infection in Eurasian wigeons and barnacle geese also showed a high level of neurotropism, as both species presented with brain lesions that co-localized with virus antigen expression. We concluded that the combination of respirotropism, neurotropism, and possibly enterotropism, contributed to the successful adaptation of 2020/2021 HPAI H5 viruses to wild waterbird populations.

Pathology and virology of natural highly pathogenic avian influenza H5N8 infection in wild Common buzzards (Buteo buteo).

Highly pathogenic avian influenza (HPAI) in wild birds is a major emerging disease, and a cause of increased mortality during outbreaks. The Common buzzard (Buteo buteo) has a considerable chance of acquiring the infection and therefore may function as bio-sentinel for the presence of virus in wildlife. This study aimed to determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection during the 2020-2021 epizootic. Eleven freshly dead, HPAI H5 virus-positive Common buzzards were necropsied. Based on RT-PCR, all birds were systemically infected with HPAI H5N8 virus, as viral RNA was detected in cloacal and pharyngeal swabs and in all 10 selected tissues of the birds, with mean Ct values per tissue ranging from 22 for heart to 32 for jejunum. Based on histology and immunohistochemistry, the most common virus-associated pathological changes were necrotizing encephalitis (9/11 birds) and necrotizing myocarditis (7/11 birds). The proventriculus of two birds showed virus-associated necrosis, indicating tropism of this virus for the digestive tract. Our advice is to collect at least a miniset of samples including brain, heart, liver, and spleen, as these tissues were positive both by RT-PCR and for virus-antigen-associated lesions.

Faeces as a novel material to estimate lyssavirus prevalence in bat populations.

Rabies is caused by infection with a lyssavirus. Bat rabies is of concern for both public health and bat conservation. The current method for lyssavirus prevalence studies in bat populations is by oral swabbing, which is invasive for the bats, dangerous for handlers, time-consuming and expensive. In many situations, such sampling is not feasible, and hence, our understanding of epidemiology of bat rabies is limited. Faeces are usually easy to collect from bat colonies without disturbing the bats and thus could be a practical and feasible material for lyssavirus prevalence studies. To further explore this idea, we performed virological analysis on faecal pellets and oral swabs of seven serotine bats (Eptesicus serotinus) that were positive for European bat 1 lyssavirus in the brain. We also performed immunohistochemical and virological analyses on digestive tract samples of these bats to determine potential sources of lyssavirus in the faeces. We found that lyssavirus detection by RT-qPCR was nearly as sensitive in faecal pellets (6/7 bats positive, 86%) as in oral swabs (7/7 bats positive, 100%). The likely source of lyssavirus in the faeces was virus excreted into the oral cavity from the salivary glands (5/6 bats positive by immunohistochemistry and RT-qPCR) or tongue (3/4 bats positive by immunohistochemistry) and swallowed with saliva. Virus could not be isolated from any of the seven faecal pellets, suggesting the lyssavirus detected in faeces is not infectious. Lyssavirus detection in the majority of faecal pellets of infected bats shows that this novel material should be further explored for lyssavirus prevalence studies in bats.

Clinical, pathological, and laboratory diagnoses of diseases of harbour porpoises (Phocoena phocoena), live stranded on the Dutch and adjacent coasts from 2003 to 2016.

Harbour porpoises (Phocoena phocoena) in the North Sea live in an environment heavily impacted by humans, the consequences of which are a concern for their health. Autopsies carried out on stranded harbour porpoises provide an opportunity to assess health problems in this species. We performed 61 autopsies on live-stranded harbour porpoises, which died following admission to a rehabilitation centre between 2003 and 2016. The animals had stranded on the Dutch (n = 52) and adjacent coasts of Belgium (n = 2) and Germany (n = 7). We assigned probable causes for stranding based on clinical and pathological criteria. Cause of stranding was associated in the majority of cases with pathologies in multiple organs (n = 29) compared to animals with pathologies in a single organ (n = 18). Our results show that the three most probable causes of stranding were pneumonia (n = 35), separation of calves from their mother (n = 10), and aspergillosis (n = 9). Pneumonia as a consequence of pulmonary nematode infection occurred in 19 animals. Pneumonia was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with Stenurus minor infection. Half of the bacterial pneumonias (6/12) could not be associated with nematode infection. Conclusions from this study are that aspergillosis is an important probable cause for stranding, while parasitic infection is not a necessary prerequisite for bacterial pneumonia, and approximately half of the animals (29/61) probably stranded due to multiple causes. An important implication of the observed high prevalence of aspergillosis is that these harbour porpoises suffered from reduced immunocompetence.

An evolutionary divergent pestivirus lacking the Npro gene systemically infects a whale species.

Pestiviruses typically infect members of the order Artiodactyla, including ruminants and pigs, although putative rat and bat pestiviruses have also been described. In the present study, we identified and characterized an evolutionary divergent pestivirus in the toothed whale species, harbour porpoise (Phocoena phocoena). We tentatively named the virus Phocoena pestivirus (PhoPeV). PhoPeV displays a typical pestivirus genome organization except for the unique absence of Npro, an N-terminal autoprotease that targets the innate host immune response. Evolutionary evidence indicates that PhoPeV emerged following an interspecies transmission event from an ancestral pestivirus that expressed Npro. We show that 9% (n = 10) of stranded porpoises from the Dutch North Sea coast (n = 112) were positive for PhoPeV and they displayed a systemic infection reminiscent of non-cytopathogenic persistent pestivirus infection. The identification of PhoPeV extends the host range of pestiviruses to cetaceans (dolphins, whales, porpoises), which are considered to have evolved from artiodactyls (even-toed ungulates). Elucidation of the pathophysiology of PhoPeV infection and Npro unique absence will add to our understanding of molecular mechanisms governing pestivirus pathogenesis.

The Canine Morbillivirus Strain Associated with An Epizootic in Caspian Seals Provides New Insights into the Evolutionary History of this Virus.

Canine morbillivirus (canine distemper virus; CDV) is a worldwide distributed morbillivirus that causes sporadic cases and recurrent epizootics among an increasing number of wild, feral, and domestic animal species. We investigated the evolutionary history of CDV strains involved in the 1988 Lake Baikal (CDVPS88) and the 2000 Caspian Sea (CDVPC00) seal die-offs by recovery of full-length sequences from archived material using next-generation sequencing. Bayesian phylogenetic analyses indicated that CDVPC00 constitutes a novel strain in a separate clade (tentatively termed "Caspian") from the America-1 clade, which is comprised of older vaccine strains. The America-1/Caspian monophyletic group is positioned most basally with respect to other clades and is estimated to have separated from other CDV clades around 1832. Our results indicate that CDVPC00 recovered from the epizootic in the Caspian Sea in 2000 belongs to a previously undetected novel clade and constitutes the most ancestral wild-type CDV clade.

Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System.

Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.IMPORTANCE Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.

Evolutionary evidence for multi-host transmission of cetacean morbillivirus.

Cetacean morbillivirus (CeMV) has emerged as the pathogen that poses the greatest risk of triggering epizootics in cetacean populations worldwide, and has a high propensity for interspecies transmission, including sporadic infection of seals. In this study, we investigated the evolutionary history of CeMV by deep sequencing wild-type viruses from tissue samples representing cetacean species with different spatiotemporal origins. Bayesian phylogeographic analysis generated an estimated evolutionary rate of 2.34 × 10-4 nucleotide substitutions/site/year and showed that CeMV evolutionary dynamics are neither host-restricted nor location-restricted. Moreover, the dolphin morbillivirus strain of CeMV has undergone purifying selection without evidence of species-specific mutations. Cell-to-cell fusion and growth kinetics assays demonstrated that CeMV can use both dolphin and seal CD150 as a cellular receptor. Thus, it appears that CeMV can readily spread among multiple cetacean populations and may pose an additional spillover risk to seals.

Beached bachelors: An extensive study on the largest recorded sperm whale Physeter macrocephalus mortality event in the North Sea.

Between the 8th January and the 25th February 2016, the largest sperm whale Physeter macrocephalus mortality event ever recorded in the North Sea occurred with 30 sperm whales stranding in five countries within six weeks. All sperm whales were immature males. Groups were stratified by size, with the smaller animals stranding in the Netherlands, and the largest in England. The majority (n = 27) of the stranded animals were necropsied and/or sampled, allowing for an international and comprehensive investigation into this mortality event. The animals were in fair to good nutritional condition and, aside from the pathologies caused by stranding, did not exhibit significant evidence of disease or trauma. Infectious agents were found, including various parasite species, several bacterial and fungal pathogens and a novel alphaherpesvirus. In nine of the sperm whales a variety of marine litter was found. However, none of these findings were considered to have been the primary cause of the stranding event. Potential anthropogenic and environmental factors that may have caused the sperm whales to enter the North Sea were assessed. Once sperm whales enter the North Sea and head south, the water becomes progressively shallower (<40 m), making this region a global hotspot for sperm whale strandings. We conclude that the reasons for sperm whales to enter the southern North Sea are the result of complex interactions of extrinsic environmental factors. As such, these large mortality events seldom have a single ultimate cause and it is only through multidisciplinary, collaborative approaches that potentially multifactorial large-scale stranding events can be effectively investigated.

Wild ducks excrete highly pathogenic avian influenza virus H5N8 (2014-2015) without clinical or pathological evidence of disease.

Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species-Eurasian wigeon (Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)-and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.

A novel cetacean adenovirus in stranded harbour porpoises from the North Sea: detection and molecular characterization.

Harbour porpoises (Phocoena phocoena) are the most prevalent cetaceans in the North Sea. The fecal viral flora of 21 harbour porpoises stranded along the Dutch coastline was analyzed by a metagenomics approach. Sequences of a novel cetacean mastadenovirus, designated harbour porpoise adenovirus 1 (HpAdV-1), were detected. The sequence of a 23-kbp genomic region, spanning the conserved late region, was determined using primer walking. Phylogenetic analysis indicated that HpAdV-1 is most closely related to bottlenose dolphin adenovirus and clusters with Cetartiodactyla adenoviruses. The prevalence of HpAdV-1 was low (2.6%) based on targeted PCR-screening of the intestinal contents of 151 harbour porpoises stranded between 2010 and 2013.

Norovirus Infection in Harbor Porpoises.

A norovirus was detected in harbor porpoises, a previously unknown host for norovirus. This norovirus had low similarity to any known norovirus. Viral RNA was detected primarily in intestinal tissue, and specific serum antibodies were detected in 8 (24%) of 34 harbor porpoises from the North Sea.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.

Vaccination Is More Effective Than Prophylactic Oseltamivir in Preventing CNS Invasion by H5N1 Virus via the Olfactory Nerve.

BACKGROUND: Influenza A viruses can replicate in the olfactory mucosa and subsequently use the olfactory nerve to enter the central nervous system (CNS). It is currently unknown whether intervention strategies are able to reduce or prevent influenza virus replication within the olfactory mucosa and subsequent spread to the CNS. Therefore, we tested the efficacy of homologous vaccination and prophylactic oseltamivir to prevent H5N1 virus CNS invasion via the olfactory nerve in our ferret model. METHODS: Ferrets were vaccinated intramuscularly or received oseltamivir (5 mg/kg twice daily) prophylactically before intranasal inoculation of highly pathogenic H5N1 virus (A/Indonesia/05/2005) and were examined using virology and pathology. RESULTS: Homologous vaccination reduced H5N1 virus replication in the olfactory mucosa and prevented subsequent virus spread to the CNS. However, prophylactic oseltamivir did not prevent H5N1 virus replication in the olfactory mucosa sufficiently, resulting in CNS invasion via the olfactory nerve causing a severe meningoencephalitis. CONCLUSIONS: Within our ferret model, vaccination is more effective than prophylactic oseltamivir in preventing CNS invasion by H5N1 virus via the olfactory nerve. This study highlights the importance of including the olfactory mucosa, olfactory nerve, and CNS tissues in future vaccine and antiviral studies, especially for viruses with a known neurotropic potential.