MicroRNAs affect GPCR and Ion channel genes needed for influenza replication.

Influenza virus causes seasonal epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses worldwide. Understanding how to regulate influenza virus replication is important for developing vaccine and therapeutic strategies. Identifying microRNAs (miRs) that affect host genes used by influenza virus for replication can support an antiviral strategy. In this study, G-protein coupled receptor (GPCR) and ion channel (IC) host genes in human alveolar epithelial (A549) cells used by influenza virus for replication (Orr-Burks et al., 2021) were examined as miR target genes following A/CA/04/09- or B/Yamagata/16/1988 replication. Thirty-three miRs were predicted to target GPCR or IC genes and their miR mimics were evaluated for their ability to decrease influenza virus replication. Paired miR inhibitors were used as an ancillary measure to confirm or not the antiviral effects of a miR mimic. Fifteen miRs lowered influenza virus replication and four miRs were found to reduce replication irrespective of virus strain and type differences. These findings provide evidence for novel miR disease intervention strategies for influenza viruses.

Drug repositioning of Clopidogrel or Triamterene to inhibit influenza virus replication in vitro.

Influenza viruses cause respiratory tract infections and substantial health concerns. Infection may result in mild to severe respiratory disease associated with morbidity and some mortality. Several anti-influenza drugs are available, but these agents target viral components and are susceptible to drug resistance. There is a need for new antiviral drug strategies that include repurposing of clinically approved drugs. Drugs that target cellular machinery necessary for influenza virus replication can provide a means for inhibiting influenza virus replication. We used RNA interference screening to identify key host cell genes required for influenza replication, and then FDA-approved drugs that could be repurposed for targeting host genes. We examined the effects of Clopidogrel and Triamterene to inhibit A/WSN/33 (EC50 5.84 uM and 31.48 uM, respectively), A/CA/04/09 (EC50 6.432 uM and 3.32 uM, respectively), and B/Yamagata/16/1988 (EC50 0.28 uM and 0.11 uM, respectively) replication. Clopidogrel and Triamterene provide a druggable approach to influenza treatment across multiple strains and subtypes.

G-Protein-Coupled Receptor and Ion Channel Genes Used by Influenza Virus for Replication.

Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza virus for replication. Targeting these proinfluenza genes can provide therapeutic strategies to reduce virus replication. Nineteen proinfluenza G-protein-coupled receptor (GPCR) and 13 proinfluenza ion channel genes were identified in human lung (A549) cells by use of small interfering RNAs (siRNAs). These proinfluenza genes were authenticated by testing influenza virus A/WSN/33-, A/CA/04/09-, and B/Yamagata/16/1988-infected A549 cells, resulting in the validation of 16 proinfluenza GPCR and 5 proinfluenza ion channel genes. These findings showed that several GPCR and ion channel genes are needed for the production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza virus productive cycle so as to limit infection.IMPORTANCE Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation, since a mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza virus during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. The targeting of host genes is refractory to drug resistance generated by viral mutations, as well as providing a platform for the development of broad-spectrum antiviral drugs.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States.

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.

Exosome-mediated human norovirus infection.

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis. Outbreaks normally occur via the fecal-oral route. HuNoV infection is thought to occur by viral particle transmission, but increasing evidence suggests a function for exosomes in HuNoV infection. HuNoV is contained within stool-derived exosomes, and exosome-associated HuNoV has been shown to replicate in human intestinal enteroids. In this study, we examine exosome-associated HuNoV infection of Vero cells and show that exosomes containing HuNoV may attach, infect, and be passaged in Vero cells. These findings support earlier findings and have implications for developing HuNoV disease intervention strategies.

Vero Cells as a Mammalian Cell Substrate for Human Norovirus.

Human norovirus (HuNoV) is a principal cause of acute gastroenteritis worldwide, particularly in developing countries. Its global prevalence is underscored by more serious morbidity and some mortality in the young (<5 years) and the elderly. To date, there are no licensed vaccines or approved therapeutics for HuNoV, mostly because there are limited cell culture systems and small animal models available. Recently described cell culture systems are not ideal substrates for HuNoV vaccine development because they are not clonal or only support a single strain. In this study, we show Vero cell-based replication of two pandemic GII.4 HuNoV strains and one GII.3 strain and confirm exosome-mediated HuNoV infection in Vero cells. Lastly, we show that trypsin addition to virus cultures or disruption of Vero cell host genes can modestly increase HuNoV replication. These data provide support for Vero cells as a cell culture model for HuNoV.

Gene-edited vero cells as rotavirus vaccine substrates.

BACKGROUND: Rotavirus (RV) is a leading cause of severe gastroenteritis globally and can cause substantial morbidity associated with gastroenteritis in children <5 years of age. Orally administered live-attenuated RV vaccines offer protection against disease but vaccination efforts have been hampered by high manufacturing costs and the need to maintain a cold chain. METHODS: A subset of Vero cell host genes was identified by siRNA that when knocked down increased RV replication and these anti-viral host genes were individually deleted using CRISPR-Cas9. RESULTS: Fully-sequenced gene knockout Vero cell substrates were assessed for increased RV replication and RV vaccine antigen expression compared to wild type Vero cells. The results showed that RV replication and antigen production were logs higher in Vero cells having an EMX2 gene deletion compared to other Vero cell substrates tested. CONCLUSIONS: We used siRNAs to screen for host genes that negatively affected RV replication, then CRISPR-Cas9 gene editing to delete select genes. The gene editing led to the development of enhanced RV vaccine substrates supporting a potential path forward for improving RV vaccine production.

Human Norovirus: Experimental Models of Infection.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. HuNoV infections lead to substantial societal and economic burdens. There are currently no licensed vaccines or therapeutics for the prevention or treatment of HuNoVs. A lack of well-characterized in vitro and in vivo infection models has limited the development of HuNoV countermeasures. Experimental infection of human volunteers and the use of related viruses such as murine NoV have provided helpful insights into HuNoV biology and vaccine and therapeutic development. There remains a need for robust animal models and reverse genetic systems to further HuNoV research. This review summarizes available HuNoV animal models and reverse genetic systems, while providing insight into their usefulness for vaccine and therapeutic development.

A universal mammalian vaccine cell line substrate.

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.