High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species. IMPORTANCE: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens.

H5N8 high-pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4B, which circulated during the 2016 epizootics in Europe, was notable for causing different clinical signs in ducks and chickens. The clinical signs preceding death were predominantly neurological in ducks versus respiratory in chickens. To investigate the determinants for the predominant neurological signs observed in ducks, we infected duck and chicken primary cortical neurons. Viral replication was identical in neuronal cultures from both species. In addition, we did not detect any major difference in the immune and inflammatory responses. These results suggest that the predominant neurological involvement of H5N8 HPAIV infection in ducks could not be recapitulated in primary neuronal cultures. In vivo, H5N8 HPAIV replication in ducks peaked soon after infection and led to an early colonization of the central nervous system. In contrast, viral replication was delayed in chickens but ultimately burst in the lungs of chickens, and the chickens died of respiratory distress before brain damage became significant. Consequently, the immune and inflammatory responses in the brain were significantly higher in duck brains than those in chickens. Our study thus suggests that early colonization of the central nervous system associated with prolonged survival after the onset of virus replication is the likely primary cause of the sustained inflammatory response and subsequent neurological disorders observed in H5N8 HPAIV-infected ducks. IMPORTANCE The severity of high-pathogenicity avian influenza virus (HPAIV) infection has been linked to its ability to replicate systemically and cause lesions in a variety of tissues. However, the symptomatology depends on the host species. The H5N8 virus of clade 2.3.4.4B had a pronounced neurotropism in ducks, leading to severe neurological disorders. In contrast, neurological signs were rarely observed in chickens, which suffered mostly from respiratory distress. Here, we investigated the determinants of H5N8 HPAIV neurotropism. We provide evidence that the difference in clinical signs was not due to a difference in neurotropism. Our results rather indicate that chickens died of respiratory distress due to intense viral replication in the lungs before viral replication in the brain could produce significant lesions. In contrast, ducks better controlled virus replication in the lungs, thus allowing the virus to replicate for a sufficient duration in the brain, to reach high levels, and to cause significant lesions.

Evolutionary and temporal dynamics of emerging influenza D virus in Europe (2009-22).

Influenza D virus (IDV) is an emerging influenza virus that was isolated for the first time in 2011 in the USA from swine with respiratory illness. Since then, IDV has been detected worldwide in different animal species, and it was also reported in humans. Molecular epidemiological studies revealed the circulation of two major clades, named D/OK and D/660. Additional divergent clades have been described but have been limited to specific geographic areas (i.e. Japan and California). In Europe, IDV was detected for the first time in France in 2012 and subsequently also in Italy, Luxembourg, Ireland, the UK, Switzerland, and Denmark. To understand the time of introduction and the evolutionary dynamics of IDV on the continent, molecular screening of bovine and swine clinical samples was carried out in different European countries, and phylogenetic analyses were performed on all available and newly generated sequences. Until recently, D/OK was the only clade detected in this area. Starting from 2019, an increase in D/660 clade detections was observed, accompanied by an increase in the overall viral genetic diversity and genetic reassortments. The time to the most recent common ancestor (tMRCA) of all existing IDV sequences was estimated as 1995-16 years before its discovery, indicating that the virus could have started its global spread in this time frame. Despite the D/OK and D/660 clades having a similar mean tMRCA (2007), the mean tMRCA for European D/OK sequences was estimated as January 2013 compared to July 2014 for European D/660 sequences. This indicated that the two clades were likely introduced on the European continent at different time points, as confirmed by virological screening findings. The mean nucleotide substitution rate of the hemagglutinin-esterase-fusion (HEF) glycoprotein segment was estimated as 1.403 × 10-3 substitutions/site/year, which is significantly higher than the one of the HEF of human influenza C virus (P < 0.0001). IDV genetic drift, the introduction of new clades on the continent, and multiple reassortment patterns shape the increasing viral diversity observed in the last years. Its elevated substitution rate, diffusion in various animal species, and the growing evidence pointing towards zoonotic potential justify continuous surveillance of this emerging influenza virus.

Influenza vaccination induces autoimmunity against orexinergic neurons in a mouse model for narcolepsy.

Narcolepsy with cataplexy or narcolepsy type 1 is a disabling chronic sleep disorder resulting from the destruction of orexinergic neurons in the hypothalamus. The tight association of narcolepsy with HLA-DQB1*06:02 strongly suggest an autoimmune origin to this disease. Furthermore, converging epidemiological studies have identified an increased incidence for narcolepsy in Europe following Pandemrix® vaccination against the 2009-2010 pandemic 'influenza' virus strain. The potential immunological link between the Pandemrix® vaccination and narcolepsy remains, however, unknown. Deciphering these mechanisms may reveal pathways potentially at play in most cases of narcolepsy. Here, we developed a mouse model allowing to track and study the T-cell response against 'influenza' virus haemagglutinin, which was selectively expressed in the orexinergic neurons as a new self-antigen. Pandemrix® vaccination in this mouse model resulted in hypothalamic inflammation and selective destruction of orexin-producing neurons. Further investigations on the relative contribution of T-cell subsets in this process revealed that haemagglutinin-specific CD4 T cells were necessary for the development of hypothalamic inflammation, but insufficient for killing orexinergic neurons. Conversely, haemagglutinin-specific CD8 T cells could not initiate inflammation but were the effectors of the destruction of orexinergic neurons. Additional studies revealed pathways potentially involved in the disease process. Notably, the interferon-γ pathway was proven essential, as interferon-γ-deficient CD8 T cells were unable to elicit the loss of orexinergic neurons. Our work demonstrates that an immunopathological process mimicking narcolepsy can be elicited by immune cross-reactivity between a vaccine antigen and a neuronal self-antigen. This process relies on a synergy between autoreactive CD4 and CD8 T cells for disease development. This work furthers our understanding of the mechanisms and pathways potentially involved in the development of a neurological side effect due to a vaccine and, likely, to narcolepsy in general.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks.

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.

Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for COVID-19 and spread rapidly following its emergence in Wuhan in 2019. Although cats are, among other domestic animals, susceptible to SARS-CoV-2 infection, little is known about their epidemiological role in the dynamics of a household infection. In this study, we monitored five cats for viral shedding daily. Each cat was confined with its COVID-19 positive owners in separate households. Low loads of viral nucleic acid were found in two cats, but only one developed anti-SARS-CoV-2 antibodies, which suggests that cats have a limited role in COVID-19 epidemiology.

Antigenic and molecular characterization of low pathogenic avian influenza A(H9N2) viruses in sub-Saharan Africa from 2017 through 2019.

Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.

Risk Mapping of Influenza D Virus Occurrence in Ruminants and Swine in Togo Using a Spatial Multicriteria Decision Analysis Approach.

Influenza D virus (IDV) has been identified in several continents, with serological evidence for the virus in Africa. In order to improve the sensitivity and cost-benefit of IDV surveillance in Togo, risk maps were drawn using a spatial multicriteria decision analysis (MCDA) and experts' opinion to evaluate the relevance of sampling areas used so far. Areas at highest risk of IDV occurrence were the main cattle markets. The maps were evaluated with previous field surveillance data collected in Togo between 2017 and 2019: 1216 sera from cattle, small ruminants, and swine were screened for antibodies to IDV by hemagglutination inhibition (HI) assays. While further samples collections are needed to validate the maps, the risk maps resulting from the spatial MCDA approach generated here highlight several priority areas for IDV circulation assessment.

Evolution of Highly Pathogenic Avian Influenza A(H5N1) Virus in Poultry, Togo, 2018.

In 2015, highly pathogenic avian influenza A(H5N1) viruses reemerged in poultry in West Africa. We describe the introduction of a reassortant clade 2.3.2.1c virus into Togo in April 2018. Our findings signal further local spread and evolution of these viruses, which could affect animal and human health.

Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections.

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.

Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production.

Ubiquitylation is one of the most versatile protein post-translational modifications and is frequently altered during virus infections. Here we employed a functional proteomics screen to identify host proteins that are differentially ubiquitylated upon dengue virus (DENV) infection. Among the several differentially modified proteins identified in infected cells was AUP1, a lipid droplet-localized type-III membrane protein, which exists predominantly in the mono-ubiquitylated form. AUP1 associated with DENV NS4A and relocalized from lipid droplets to autophagosomes upon infection. Virus production was abolished in cells deleted for AUP1 or expressing an AUP1 acyltransferase domain mutant. Ubiquitylation disrupted the AUP1-NS4A interaction, resulting in inhibited acyltransferase activity, defective lipophagy, and attenuated virus production. Our results show that DENV-NS4A exploits the acyltransferase activity of AUP1 to trigger lipophagy, a process regulated by ubiquitylation. This mechanism appears to be a general phenomenon in biogenesis of flaviviruses and underscores the critical role of post-translational modifications in virus infections.