Primary harbour seal (Phoca vitulina) airway epithelial cells show high susceptibility to infection by a seal-derived influenza A virus (H5N8).

Highly pathogenic avian influenza viruses of the H5N8 subtype have been circulating in Europe and Asia since 2016, causing huge economic losses to the poultry industry. A new wave of H5Nx infections has begun in 2020. The viruses mainly infect wild birds and waterfowl; from there they spread to poultry and cause diseases. Previous studies have shown that the H5N8 viruses have seldom spread to mammals; however, reports in early 2021 indicate that humans may be infected, and some incident reports indicate that H5Nx clade 2.3.4.4B virus may be transmitted to wild mammals, such as red foxes and seals. In order to get more information on how the H5N8 virus affects seals and other marine animals, here, we used primary cultures to analyze the cell tropism of the H5N8 virus, which was isolated from an infected grey seal (H5N8/Seal-2016). Primary tracheal epithelial cells were readily infected by H5N8/Seal -2016 virus; in contrast, the commonly used primary seal kidney cells required the presence of exogenous trypsin to initiate virus infection. When applied to an ex vivo precision-cut lung slice model, compared with recombinant human H3N2 virus or H9N2 LPAI virus, the H5N8/Seal-2016 virus replicated to a high titre and caused a strong detrimental effect; with these characteristics, the virus was superior to a human H3N2 virus and to an H9N2 LPAI virus. By using well-differentiated air-liquid interface (ALI) cultures, we have observed that ALI cultures of canines, ferrets, and harbour seals are more sensitive to H5N8/Seal-2016 virus than are human or porcine ALI cultures, which cannot be fully explained by sialic acid distribution. Our results indicate that the airway epithelium of carnivores may be the main target of H5N8 viruses. Consideration should be given to an increased monitoring of the distribution of highly pathogenic avian influenza viruses in wild animals.

Overcoming the Barrier of the Respiratory Epithelium during Canine Distemper Virus Infection.

Canine distemper virus (CDV) is a highly contagious pathogen and is known to enter the host via the respiratory tract and disseminate to various organs. Current hypotheses speculate that CDV uses the homologous cellular receptors of measles virus (MeV), SLAM and nectin-4, to initiate the infection process. For validation, here, we established the well-differentiated air-liquid interface (ALI) culture model from primary canine tracheal airway epithelial cells. By applying the green fluorescent protein (GFP)-expressing CDV vaccine strain and recombinant wild-type viruses, we show that cell-free virus infects the airway epithelium mainly via the paracellular route and only after prior disruption of tight junctions by pretreatment with EGTA; this infection was related to nectin-4 but not to SLAM. Remarkably, when CDV-preinfected DH82 cells were cocultured on the basolateral side of canine ALI cultures grown on filter supports with a 1.0-µm pore size, cell-associated CDV could be transmitted via cell-to-cell contact from immunocytes to airway epithelial cultures. Finally, we observed that canine ALI cultures formed syncytia and started to release cell-free infectious viral particles from the apical surface following treatment with an inhibitor of the JAK/STAT signaling pathway (ruxolitinib). Our findings show that CDV can overcome the epithelial barrier through different strategies, including infection via immunocyte-mediated transmission and direct infection via the paracellular route when tight junctions are disrupted. Our established model can be adapted to other animals for studying the transmission routes and the pathogenicity of other morbilliviruses. IMPORTANCE Canine distemper virus (CDV) is not only an important pathogen of carnivores, but it also serves as a model virus for analyzing measles virus pathogenesis. To get a better picture of the different stages of infection, we used air-liquid interface cultures to analyze the infection of well-differentiated airway epithelial cells by CDV. Applying a coculture approach with DH82 cells, we demonstrated that cell-mediated infection from the basolateral side of well-differentiated epithelial cells is more efficient than infection via cell-free virus. In fact, free virus was unable to infect intact polarized cells. When tight junctions were interrupted by treatment with EGTA, cells became susceptible to infection, with nectin-4 serving as a receptor. Another interesting feature of CDV infection is that infection of well-differentiated airway epithelial cells does not result in virus egress. Cell-free virions are released from the cells only in the presence of an inhibitor of the JAK/STAT signaling pathway. Our results provide new insights into how CDV can overcome the barrier of the airway epithelium and reveal similarities and some dissimilarities compared to measles virus.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

Avian Influenza A Virus Infects Swine Airway Epithelial Cells without Prior Adaptation.

Pigs play an important role in the interspecies transmission of influenza A viruses (IAV). The porcine airway epithelium contains binding sites for both swine/human IAV (α2,6-linked sialic acids) and avian IAV (α2,3-linked sialic acids) and therefore is suited for adaptation of viruses from other species as suggested by the "mixing vessel theory". Here, we applied well-differentiated swine airway epithelial cells to find out whether efficient infection by avian IAV requires prior adaption. Furthermore, we analyzed the influence of the sialic acid-binding activity and the virus-induced detrimental effects. Surprisingly, an avian IAV H1N1 strain circulating in European poultry and waterfowl shows increased and prolonged viral replication without inducing a strong innate immune response. This virus could infect the lower respiratory tract in our precision cut-lung slice model. Pretreating the cells with poly (I:C) and/or JAK/STAT pathway inhibitors revealed that the interferon-stimulated innate immune response influences the replication of avian IAV in swine airway epitheliums but not that of swine IAV. Further studies indicated that in the infection by IAVs, the binding affinity of sialic acid is not the sole factor affecting the virus infectivity for swine or human airway epithelial cells, whereas it may be crucial in well-differentiated ferret tracheal epithelial cells. Taken together, our results suggest that the role of pigs being the vessel of interspecies transmission should be reconsidered, and the potential of avian H1N1 viruses to infect mammals needs to be characterized in more detail.

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

Highly Pathogenic Avian Influenza A(H5N8) Virus in Gray Seals, Baltic Sea.

We detected a highly pathogenic avian influenza A(H5N8) virus in lung samples of 2 gray seals (Halichoerus grypus) stranded on the Baltic coast of Poland in 2016 and 2017. This virus, clade 2.3.4.4 B, was closely related to avian H5N8 viruses circulating in Europe at the time.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions.

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

Infection Studies in Pigs and Porcine Airway Epithelial Cells Reveal an Evolution of A(H1N1)pdm09 Influenza A Viruses Toward Lower Virulence.

We analyzed the virulence of pandemic H1N1 2009 influenza A viruses in vivo and in vitro. Selected viruses isolated in 2009, 2010, 2014, and 2015 were assessed using an aerosol-mediated high-dose infection model for pigs as well as air-liquid interface cultures of differentiated airway epithelial cells. Using a dyspnea score, rectal temperature, lung lesions, and viral load in the lung as parameters, the strains from 2014-2015 were significantly less virulent than the strains isolated in 2009-2010. In vitro, the viruses from 2009-2010 also differed from the 2014-2015 viruses by increased release of infectious virus, a more pronounced loss of ciliated cells, and a reduced thickness of the epithelial cell layer. Our in vivo and in vitro results reveal an evolution of A(H1N1)pdm09 viruses toward lower virulence. Our in vitro culture system can be used to predict the virulence of influenza viruses.

Sialic acid-dependent interaction of group B streptococci with influenza virus-infected cells reveals a novel adherence and invasion mechanism.

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3-linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3-linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co-sedimentation assay revealed that viruses with a preference for α2,3-linked sialic acids bind to GBS in a sialic acid-dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination-inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre-infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co-infected by influenza viruses. This co-infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3-linked sialic acids and human viruses binding to this linkage type.

Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells.

We analyzed the adaptation of influenza viruses to growth in differentiated airway epithelial cells of a new host by passaging an avian H9N2 virus three times in porcine precision-cut lung slices (PCLS). Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins, and two in the HA protein. In this study, we characterized the PB2 mutation G685R by generating recombinant H9N2 viruses containing the PB2 single mutation alone or in combination with one of the HA mutations (A190V or T212I). When analyzed in porcine cells - a tracheal cell line (NPTr) or PCLS - the PB2-685 mutant did not provide a growth advantage and had no effect on the ciliary activity which is a virulence marker of swine influenza viruses. Pathogenicity for mice was also not increased by the single PB2 mutation. However, both double mutants (HA-190+PB2-685 and HA-212+PB2-685) showed significantly increased virulence in mice. Therefore, the mutations in the HA and PB2 proteins may confer early adaptation of an avian H9N2 virus to a mammalian host. In conclusion, we expect that a broader ensemble of mutations will be required to render an H9N2 virus virulent for pigs.

The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells.

Virus-host interactions in the respiratory epithelium during long term influenza virus infection are not well characterized. Therefore, we developed an air-liquid interface culture system for differentiated porcine respiratory epithelial cells to study the effect of virus-induced cellular damage. In our well-differentiated cells, α2,6-linked sialic acid is predominantly expressed on the apical surface and the basal cells mainly express α2,3-linked sialic acid. During the whole infection period, release of infectious virus was maintained at a high titre for more than seven days. The infected epithelial cells were subject to apoptosis resulting in the loss of ciliated cells together with a thinner thickness. Nevertheless, the airway epithelium maintained trans-epithelial electrical resistance and retained its barrier function. The loss of ciliated cells was compensated by the cells which contained the KRT5 basal cell marker but were not yet differentiated into ciliated cells. These specialized cells showed an increase of α2,3-linked sialic acid on the apical surface. In sum, our results help to explain the localized infection of the airway epithelium by influenza viruses. The impairment of mucociliary clearance in the epithelial cells provides an explanation why prior viral infection renders the host more susceptible to secondary co-infection by another pathogen.

Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions.

Streptococci may colonize the epithelium in the airways and other entry sites. While local infection often remains asymptomatic, severe or even fatal diseases occur when streptococci become invasive and spread to different sites in the infected host. We have established porcine respiratory air-liquid interface cultures (ALI) from the porcine lung to analyze the interaction of streptococci with their primary target cells. As representative of the streptococcal family we chose Streptococcus suis (S. suis) that is not only a major swine respiratory pathogen but can also infect humans. Suilysin, a cholesterol-dependent cytolysin (CDC), is an important virulence factor. By comparing a S. suis wt strain with a suilysin-deficient mutant, we demonstrate that suilysin contributes to (i) adherence to airway cells (ii) loss of ciliated cells (iii) apoptosis, and (iv) invasion. Furthermore, we show that cytolytic activity of suilysin is crucial for these effects. A striking result of our analysis was the high efficiency of S. suis-induced apoptosis and invasion upon infection under ALI conditions. These properties have been reported to be less efficient when analyzed with immortalized cells. We hypothesize that soluble effectors such as suilysin are present at higher concentrations in cells kept at ALI conditions and thus more effective. These results should be relevant also for infection of the respiratory tract by other respiratory pathogens.

Sialic acid-dependent interactions between influenza viruses and Streptococcus suis affect the infection of porcine tracheal cells.

Bacterial co-infections are a major complication in influenza-virus-induced disease in both humans and animals. Either of the pathogens may induce a host response that affects the infection by the other pathogen. A unique feature in the co-infection by swine influenza viruses (SIV) and Streptococcus suis serotype 2 is the direct interaction between the two pathogens. It is mediated by the haemagglutinin of SIV that recognizes the α2,6-linked sialic acid present in the capsular polysaccharide of Streptococcus suis. In the present study, this interaction was demonstrated for SIV of both H1N1 and H3N2 subtypes as well as for human influenza viruses that recognize α2,6-linked sialic acid. Binding of SIV to Streptococcus suis resulted in co-sedimentation of virus with bacteria during low-speed centrifugation. Viruses bound to bacteria retained infectivity but induced only tiny plaques compared with control virus. Infection of porcine tracheal cells by SIV facilitated adherence of Streptococcus suis, which was evident by co-staining of bacterial and viral antigen. Sialic-acid-dependent binding of Streptococcus suis was already detectable after incubation for 30 min. By contrast, bacterial co-infection had a negative effect on the replication of SIV as indicated by lower virus titres in the supernatant and a delay in the kinetics of virus release.

Detection of anti-reticuloendotheliosis virus antibody by blocking enzyme-linked immunosorbent assay with expression envelope protein.

The current reticuloendotheliosis virus (REV) antibody detection kit that uses enzyme-linked immunosorbent assay (ELISA) needs concentrated virus, which is difficult to obtain due to its poor propagation in cells. In addition, this kit detects only chicken antibody but not other species. To overcome these disadvantages, we cloned and expressed REV env gene to develop monoclonal antibodies (mAbs), which we used for antibody detection in ELISA. Three mAbs were prepared from mice. These three mAbs could recognize REVs from ducks and geese by immunodot assay. In addition, the epitopes that the three mAbs recognized were determined by using three different env protein fragments by western blotting. One mAb was used to develop a blocking ELISA (bELISA) coated with expressed env protein to detect anti-REV antibody in chicken serum. This assay had a 98.8% (79/80) agreement with a commercial ELISA kit. Another 146 chicken sera with known neutralization antibodies were used as positive controls to evaluate this bELISA. The sensitivity and specificity this bELISA were 88.9% (40/45) and 94.8% (91/96), respectively. Thus, this bELISA could be used for anti-REV antibody detection in birds.