MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.

Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid.

In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the α-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. IMPORTANCE Mumps virus (MuV) infection may cause serious diseases, including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative-strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.

A Single Dose of an Avian H3N8 Influenza Virus Vaccine Is Highly Immunogenic and Efficacious against a Recently Emerged Seal Influenza Virus in Mice and Ferrets.

UNLABELLED: H3N8 influenza viruses are a commonly found subtype in wild birds, usually causing mild or no disease in infected birds. However, they have crossed the species barrier and have been associated with outbreaks in dogs, pigs, donkeys, and seals and therefore pose a threat to humans. A live attenuated, cold-adapted (ca) H3N8 vaccine virus was generated by reverse genetics using the wild-type (wt) hemagglutinin (HA) and neuraminidase (NA) genes from the A/blue-winged teal/Texas/Sg-00079/2007 (H3N8) (tl/TX/079/07) wt virus and the six internal protein gene segments from the ca influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (H2N2), the backbone of the licensed seasonal live attenuated influenza vaccine. One dose of the tl/TX/079/07 ca vaccine induced a robust neutralizing antibody response against the homologous (tl/TX/079/07) and two heterologous influenza viruses, including the recently emerged A/harbor seal/New Hampshire/179629/2011 (H3N8) and A/northern pintail/Alaska/44228-129/2006 (H3N8) viruses, and conferred robust protection against the homologous and heterologous influenza viruses. We also analyzed human sera against the tl/TX/079/07 H3N8 avian influenza virus and observed low but detectable antibody reactivity in elderly subjects, suggesting that older H3N2 influenza viruses confer some cross-reactive antibody. The latter observation was confirmed in a ferret study. The safety, immunogenicity, and efficacy of the tl/TX/079/07 ca vaccine in mice and ferrets support further evaluation of this vaccine in humans for use in the event of transmission of an H3N8 avian influenza virus to humans. The human and ferret serology data suggest that a single dose of the vaccine may be sufficient in older subjects. IMPORTANCE: Although natural infection of humans with an avian H3N8 influenza virus has not yet been reported, this influenza virus subtype has already crossed the species barrier and productively infected mammals. Pandemic preparedness is an important public health priority. Therefore, we generated a live attenuated avian H3N8 vaccine candidate and demonstrated that a single dose of the vaccine was highly immunogenic and protected mice and ferrets against homologous and heterologous H3N8 avian viruses.

Influenza H1N1pdm-specific maternal antibodies offer limited protection against wild-type virus replication and influence influenza vaccination in ferrets.

OBJECTIVE: The objective was to study passively acquired influenza H1N1 pandemic (H1N1pdm) maternal antibody kinetics and its impact on subsequent influenza infection and vaccination in ferrets during an outbreak of the H1N1pdm. DESIGN AND MAIN OUTCOME MEASURES: Infectivity of the H1N1pdm in the respiratory tract of ferrets was compared with the previous seasonal A/South Dakota/6/2007 (SD07, H1N1). Influenza-specific antibodies were quantitated and antibody-mediated protection against the homologous and heterologous H1N1 virus challenge infection was determined. RESULTS: H1N1pdm virus was approximately 10 times more infectious than SD07 in ferrets, replicated to higher viral titers in the upper respiratory tract and shed for a longer duration. Influenza-specific antibodies after natural infection persisted much longer in the circulation than passively acquired maternal antibodies. The protection conferred by the maternal antibodies was limited to the homologous virus strain and was ineffective against SD07 and H3N2 virus. Serum antibodies from maternal transmission or passive transfer interfered with homologous vaccine strain-mediated antibody responses in the ferret. A booster immunization was required to elicit a high level of antibody. CONCLUSIONS: The findings support the rationale for a prime and boost immunization strategy in young children in whom maternal antibodies are present.

Evaluation of the humoral and cellular immune responses elicited by the live attenuated and inactivated influenza vaccines and their roles in heterologous protection in ferrets.

The humoral and cellular immune responses elicited by the trivalent live attenuated influenza vaccine (LAIV) and the trivalent inactivated influenza vaccine (TIV) were evaluated in the ferret model, using newly developed ferret immunological reagents and assays. In contrast to the TIV, which only induced immune responses in primed animals, LAIV induced strong influenza virus-specific serum antibody and T-cell responses in both naive and influenza-seropositive animals. The LAIV offered significant protection against a heterologous H1N1 virus challenge infection in the upper respiratory tract. Influenza virus-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody-secreting cells (ASCs) and influenza virus-specific CD4(+) and CD8(+) T cells were detected in the circulation and local paratracheal draining lymph nodes. The frequency of the influenza-specific ASCs in the local lymph nodes appeared to correlate with the degree of protection in the upper respiratory tract. The protection conferred by the LAIV could be attributed not only to the antibody response but also to the cell-mediated and local mucosal immune responses, particularly in naive ferrets. These findings may explain why the LAIV is immunologically superior and offers immediate protection after a single dose in children.

Surface glycoproteins of influenza A H3N2 virus modulate virus replication in the respiratory tract of ferrets.

The hemagglutinin (HA) genes of the influenza A H3N2 subtype viruses isolated from 1968 to 2010 have evolved substantially but their neuraminidase (NA) genes have been relatively less divergent. The H3N2 viruses isolated since 1995 were found to replicate in the lower respiratory tract of ferrets less efficiently than the earlier isolates. To evaluate whether the HA or/and NA or the internal protein gene segments of the H3N2 virus affected viral replication in the respiratory tract of ferrets, recombinant A/California/07/2004 (CA04) (H3N2) virus and its reassortants that contained the same CA04 internal protein gene segments and the HA and/or NA of A/Udorn/309/1972 (UD72) or A/Wuhan/359/1995 (WH95) H3N2 viruses were generated and evaluated for their replication in the respiratory tract of ferrets. All the reassortant viruses replicated efficiently in the upper respiratory tract of ferrets, but their replication in the lower respiratory tract of ferrets varied. In contrast to the UD72-HA reassortant virus that replicated efficiently in the lungs of ferrets, the virus with the WH95-HA or the CA04-HA either replicated modestly or did not replicate in the lungs of ferrets. The reassortants with the WH95-HA and UD72-NA or CA04-NA had the tendency to lose a N-linked glycosylation site at residue 246 in the HA, resulting in viral lung titer of 100-fold higher than the virus with the HA and NA from WH95. The UD72-NA had the highest neuraminidase activity and increased viral replication by up to 100-fold in tissue culture cells during early infection. Thus, our data indicate that both the HA and NA glycoproteins play important roles in viral replication of the H3N2 influenza virus in ferrets.

Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350.

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1(st) or the 3(rd) position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3(rd) position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation.