A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

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.

Effect of 2020-21 and 2021-22 Highly Pathogenic Avian Influenza H5 Epidemics on Wild Birds, the Netherlands.

The number of highly pathogenic avian influenza (HPAI) H5-related infections and deaths of wild birds in Europe was high during October 1, 2020-September 30, 2022. To quantify deaths among wild species groups with known susceptibility for HPAI H5 during those epidemics, we collected and recorded mortality data of wild birds in the Netherlands. HPAI virus infection was reported in 51 bird species. The species with the highest numbers of reported dead and infected birds varied per epidemic year; in 2020-21, they were within the Anatidae family, in particular barnacle geese (Branta leucopsis) and in 2021-22, they were within the sea bird group, particularly Sandwich terns (Thalasseus sandvicensis) and northern gannet (Morus bassanus). Because of the difficulty of anticipating and modeling the future trends of HPAI among wild birds, we recommend monitoring live and dead wild birds as a tool for surveillance of the changing dynamics of HPAI.

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.

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.

Increase of mast cells in COVID-19 pneumonia may contribute to pulmonary fibrosis and thrombosis.

AIMS: Lung tissue from COVID-19 patients shares similar histomorphological features with chronic lung allograft disease, also suggesting activation of autoimmune-related pathways in COVID-19. To more clearly understand the underlying spectrum of pathophysiology in COVID-19 pneumonia, we analysed mRNA expression of autoimmune-related genes in post-mortem lung tissue from COVID-19 patients. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded lung tissue samples of 18 COVID-19 patients and eight influenza patients were used for targeted gene expression profiling using NanoString technology. Multiplex immunofluorescence for tryptase and chymase was applied for validation. Genes related to mast cells were significantly increased in COVID-19. This finding was strengthened by multiplex immunofluorescence also showing a significant increase of tryptase- and chymase-positive cells in COVID-19. Furthermore, receptors for advanced glycation end-products (RAGE) and pro-platelet basic protein (PPBP) were up-regulated in COVID-19 compared to influenza. Genes associated with Type I interferon signalling showed a significant correlation to detected SARS-CoV2 pathway-related genes. The comparison of lung tissue samples from both groups based on the presence of histomorphological features indicative of acute respiratory distress syndrome did not result in finding any specific gene or pathways. CONCLUSION: Two separate means of measuring show a significant increase of mast cells in SARS-CoV-2-infected lung tissue compared to influenza. Additionally, several genes involved in fibrosis and thrombosis, among which are RAGE and PPBP, are up-regulated in COVID-19. As mast cells are able to induce thrombosis and fibrosis, they may play an important role in the pathogenesis of COVID-19.

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.

Genesis and spread of multiple reassortants during the 2016/2017 H5 avian influenza epidemic in Eurasia.

Highly pathogenic avian influenza (HPAI) viruses of the H5 A/goose/Guangdong/1/96 lineage can cause severe disease in poultry and wild birds, and occasionally in humans. In recent years, H5 HPAI viruses of this lineage infecting poultry in Asia have spilled over into wild birds and spread via bird migration to countries in Europe, Africa, and North America. In 2016/2017, this spillover resulted in the largest HPAI epidemic on record in Europe and was associated with an unusually high frequency of reassortments between H5 HPAI viruses and cocirculating low-pathogenic avian influenza viruses. Here, we show that the seven main H5 reassortant viruses had various combinations of gene segments 1, 2, 3, 5, and 6. Using detailed time-resolved phylogenetic analysis, most of these gene segments likely originated from wild birds and at dates and locations that corresponded to their hosts' migratory cycles. However, some gene segments in two reassortant viruses likely originated from domestic anseriforms, either in spring 2016 in east China or in autumn 2016 in central Europe. Our results demonstrate that, in addition to domestic anseriforms in Asia, both migratory wild birds and domestic anseriforms in Europe are relevant sources of gene segments for recent reassortant H5 HPAI viruses. The ease with which these H5 HPAI viruses reassort, in combination with repeated spillovers of H5 HPAI viruses into wild birds, increases the risk of emergence of a reassortant virus that persists in wild bird populations yet remains highly pathogenic for poultry.

Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

Host Range of Influenza A Virus H1 to H16 in Eurasian Ducks Based on Tissue and Receptor Binding Studies.

Dabbling and diving ducks partly occupy shared habitats but have been reported to play different roles in wildlife infectious disease dynamics. Influenza A virus (IAV) epidemiology in wild birds has been based primarily on surveillance programs focused on dabbling duck species, particularly mallard (Anas platyrhynchos). Surveillance in Eurasia has shown that in mallards, some subtypes are commonly (H1 to H7 and H10), intermediately (H8, H9, H11, and H12), or rarely (H13 to H16) detected, contributing to discussions on virus host range and reservoir competence. An alternative to surveillance in determining IAV host range is to study virus attachment as a determinant for infection. Here, we investigated the attachment patterns of all avian IAV subtypes (H1 to H16) to the respiratory and intestinal tracts of four dabbling duck species (Mareca and Anas spp.), two diving duck species (Aythya spp.), and chicken, as well as to a panel of 65 synthetic glycan structures. We found that IAV subtypes generally showed abundant attachment to colon of the Anas duck species, mallard, and Eurasian teal (Anas crecca), supporting the fecal-oral transmission route in these species. The reported glycan attachment profile did not explain the virus attachment patterns to tissues but showed significant attachment of duck-originated viruses to fucosylated glycan structures and H7 virus tropism for Neu5Gc-LN. Our results suggest that Anas ducks play an important role in the ecology and epidemiology of IAV. Further knowledge on virus tissue attachment, receptor distribution, and receptor binding specificity is necessary to understand the mechanisms underlying host range and epidemiology of IAV.IMPORTANCE Influenza A viruses (IAVs) circulate in wild birds worldwide. From wild birds, the viruses can cause outbreaks in poultry and sporadically and indirectly infect humans. A high IAV diversity has been found in mallards (Anas platyrhynchos), which are most often sampled as part of surveillance programs; meanwhile, little is known about the role of other duck species in IAV ecology and epidemiology. In this study, we investigated the attachment of all avian IAV hemagglutinin (HA) subtypes (H1 to H16) to tissues of six different duck species and chicken as an indicator of virus host range. We demonstrated that the observed virus attachment patterns partially explained reported field prevalence. This study demonstrates that dabbling ducks of the Anas genus are potential hosts for most IAV subtypes, including those infecting poultry. This knowledge is useful to target the sampling of wild birds in nature and to further study the interaction between IAVs and birds.

Measles skin rash: Infection of lymphoid and myeloid cells in the dermis precedes viral dissemination to the epidermis.

Measles is characterized by fever and a maculopapular skin rash, which is accompanied by immune clearance of measles virus (MV)-infected cells. Histopathological analyses of skin biopsies from humans and non-human primates (NHPs) with measles rash have identified MV-infected keratinocytes and mononuclear cells in the epidermis, around hair follicles and near sebaceous glands. Here, we address the pathogenesis of measles skin rash by combining data from experimentally infected NHPs, ex vivo infection of human skin sheets and in vitro infection of primary human keratinocytes. Analysis of NHP skin samples collected at different time points following MV inoculation demonstrated that infection in the skin precedes onset of rash by several days. MV infection was detected in lymphoid and myeloid cells in the dermis before dissemination to the epidermal leukocytes and keratinocytes. These data were in good concordance with ex vivo MV infections of human skin sheets, in which dermal cells were more targeted than the epidermal cells. To address viral dissemination to the epidermis and to determine whether the dissemination is receptor-dependent, we performed experimental infections of primary keratinocytes collected from healthy donors. These experiments demonstrated that MV infection of keratinocytes is mainly nectin-4-dependent, and differentiated keratinocytes, which express higher levels of nectin-4, are more susceptible to MV infection than proliferating keratinocytes. Based on these data, we propose a model to explain measles skin rash: migrating MV-infected lymphocytes initiate the infection of dermal skin-resident CD150+ immune cells. The infection is subsequently disseminated from the dermal papillae to nectin-4+ keratinocytes in the basal epidermis. Lateral spread of MV infection is observed in the superficial epidermis, most likely due to the higher level of nectin-4 expression on differentiated keratinocytes. Finally, MV-infected cells are cleared by infiltrating immune cells, causing hyperemia and edema, which give the appearance of morbilliform skin rash.

Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses.

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.

HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS (H5N8) OUTBREAK IN A WILD BIRD RESCUE CENTER, THE NETHERLANDS: CONSEQUENCES AND RECOMMENDATIONS.

Since the emergence of the Goose/Guangdong H5 lineage in 1996 and spillover of highly pathogenic avian influenza (HPAI) from poultry to wild birds, outbreaks have become increasingly frequent in wild birds. The latest outbreak in the Netherlands occurred in the fall-winter of 2020-2021 and was linked to incursions of HPAI H5N8 virus. During the larger national outbreak, wild birds in rehabilitation center "Vogelklas Karel Schot (VKS)" in Rotterdam presented with clinical signs compatible with HPAI, including head shaking, torticollis, and abnormal gait. During an epidemiologic investigation at VKS, water samples from the pools in the enclosures and oropharyngeal and cloacal swabs from 128 birds of different species were analyzed for the presence of H5N8 virus. Forty-five birds and the pool water tested positive for the virus. The outbreak at VKS was likely introduced by one or more infected geese (Anser anser, Anser anser domesticus, Branta leucopsis), after which the virus spread via pool water and with the relocation of infected birds within the center. In principle, such outbreaks are preventable. Recent updates about HPAI to provide guidance to help avoid future incursions of HPAI into wildlife rescue centers are reported.

SARS-CoV-2 Neutralizing Human Antibodies Protect Against Lower Respiratory Tract Disease in a Hamster Model.

Effective clinical intervention strategies for coronavirus disease 2019 (COVID-19) are urgently needed. Although several clinical trials have evaluated use of convalescent plasma containing virus-neutralizing antibodies, levels of neutralizing antibodies are usually not assessed and the effectiveness has not been proven. We show that hamsters treated prophylactically with a 1:2560 titer of human convalescent plasma or a 1:5260 titer of monoclonal antibody were protected against weight loss, had a significant reduction of virus replication in the lungs, and showed reduced pneumonia. Interestingly, this protective effect was lost with a titer of 1:320 of convalescent plasma. These data highlight the importance of screening plasma donors for high levels of neutralizing antibodies. Our data show that prophylactic administration of high levels of neutralizing antibody, either monoclonal or from convalescent plasma, prevent severe SARS-CoV-2 pneumonia in a hamster model, and could be used as an alternative or complementary to other antiviral treatments for COVID-19.

Reverse Zoonosis of COVID-19: Lessons From the 2009 Influenza Pandemic.

Over the past decade, pandemics caused by pandemic H1N1 (pH1N1) influenza virus in 2009 and severe acute respiratory syndrome virus type 2 (SARS-CoV-2) in 2019 have emerged. Both are high-impact respiratory pathogens originating from animals. Their wide distribution in the human population subsequently results in an increased risk of human-to-animal transmission: reverse zoonosis. Although there have only been rare reports of reverse zoonosis events associated with the ongoing coronavirus disease 2019 (COVID-19) pandemic from SARS-CoV-2 so far, comparison with the pH1N1 influenza pandemic can provide a better understanding of the possible consequences of such events for public and animal health. The results of our review suggest that similar factors contribute to successful crossing of the host species barriers in both pandemics. Specific risk factors include sufficient interaction between infected humans and recipient animals, suitability of the animal host factors for productive virus infection, and suitability of the animal host population for viral persistence. Of particular concern is virus spread to susceptible animal species, in which group housing and contact network structure could potentially result in an alternative virus reservoir, from which reintroduction into humans can take place. Virus exposure in high-density populations could allow sustained transmission in susceptible animal species. Identification of the risk factors and serological surveillance in SARS-CoV-2-susceptible animal species that are group-housed should help reduce the threat from reverse zoonosis of COVID-19.

In vivo comparison of a laboratory-adapted and clinical-isolate-based recombinant human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract disease in infants. Most HRSV infections remain restricted to the upper respiratory tract (URT), but in a small percentage of patients the infection spreads to the lower respiratory tract, resulting in bronchiolitis or pneumonia. We have a limited understanding of HRSV pathogenesis and what factors determine disease severity, partly due to the widespread use of tissue-culture-adapted viruses. Here, we studied early viral dissemination and tropism of HRSV in cotton rats, BALB/cJ mice and C57BL/6 mice. We used a novel recombinant (r) strain based on a subgroup A clinical isolate (A11) expressing EGFP [rHRSVA11EGFP(5)]. A recombinant laboratory-adapted HRSV strain [rHRSVA2EGFP(5)] was used as a direct comparison. Our results show that rHRSVA11EGFP(5) replicated to higher viral titres than laboratory-adapted rHRSVA2EGFP(5) in the URT of cotton rats and mice. HRSV-infected cells were detected as early as 2 days post-inoculation in both species in the nasal septa and lungs. Infection was predominantly present in ciliated epithelial cells in cotton rats and in the olfactory mucosa of mice. In our opinion, this study highlights that the choice of virus strain is important when studying HRSV pathogenesis in vivo and demonstrates that A11 is a representative clinical-based virus. Additionally, we show critical differences in tropism and inflammation when comparing HRSV infection of cotton rats and mice.

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.

Enterotropism of highly pathogenic avian influenza virus H5N8 from the 2016/2017 epidemic in some wild bird species.

In 2016/2017, H5N8 highly pathogenic avian influenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian influenza (LPAI) viruses, allowing more efficient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in respiratory epithelium, and one species only in intestinal epithelium. Virus antigen expression was association with inflammation and necrosis in multiple tissues. The level of attachment to wild duck intestinal epithelia of 2016 H5N8 virus was comparable to that of LPAI H4N5 virus, and higher than that of 2005 H5N1 virus for two of the four duck species and chicken tested. Overall, these results indicate that 2016 H5N8 may have acquired a similar enterotropism to LPAI viruses, without having lost the respirotropism of older HPAI viruses of the Goose/Guangdong lineage. The increased enterotropism of 2016 H5N8 implies that this virus had an increased chance to persist long term in the wild waterbird reservoir.