Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018.

ABSTRACTLow pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures.

Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.

Stable incorporation of GM-CSF into dissolvable microneedle patch improves skin vaccination against influenza.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells.

UNLABELLED: Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-ΔgDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV-ΔgDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-ΔgDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-ΔgDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. IMPORTANCE: B virus is the only known simplexvirus that causes zoonotic infection, resulting in approximately 80% mortality in untreated humans or in lifelong persistence with the constant threat of reactivation in survivors. Here, we report that B virus lacking the gD envelope glycoprotein infects both human and monkey cells as efficiently as wild-type B virus. These data provide evidence for a novel mechanism(s) utilized by B virus to gain access to target cells. This mechanism is different from those used by its close relatives, HSV-1 and -2, where gD is a pivotal protein in the virus entry process. The possibility remains that unidentified receptors, specific for B virus, permit virus entry into target cells through gD-independent pathways. Understanding the molecular mechanisms of B virus entry may help in developing rational therapeutic strategies for the prevention and treatment of B virus infection in both macaques and humans.