Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins

Abstract Background Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies. Methods In this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes. Results Seventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B). Conclusions Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.http://deepblue.lib.umich.edu/bitstream/2027.42/110223/1/12985_2014_Article_228.pd

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo including infectious material between cells

We discovered that cells expressing the adherens junction protein nectin-1 capture nectin-4 containing membranes from the surface of adjacent cells in a trans-endocytosis process. Internalized nectin-1/4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1 expressing cells acquire dye-labelled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism.</jats:p

Humidity as a non-pharmaceutical intervention for influenza A

AbstractInfluenza is a global problem infecting 5-10 % of adults and 20-30 % of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza and its amount. Additionally samples were tested for their ability to infect cells in cultures. Deliberate classroom humidification (with commercial steam humidifiers) resulted in a significant reduction of the total number of influenza positive samples (air and fomite), viral copy number, and efficiency of viral infectivity. This is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks.Author summaryHuman influenza infections have a substantial impact on society (including lost productivity and medical costs). Children, 3-4 years of age are the main introducers and spreaders of influenza within a household and community. There is evidence from laboratory and epidemiological studies that suggests that low humidity in winter (in temperate climates) may increase the ability of influenza virus to survive and spread between individuals. We wanted to know if added in humidity (through steam humidifiers) could reduce the amount of influenza present and its spread within preschool classrooms (students aged 3-4 years)? Additionally, we looked at the infectivity of the influenza isolated and if there were differences in the number of students with influenza-like illnesses during our study. We show that humidification can reduce the amount of influenza present within samples from preschool classrooms and that there were fewer infectious samples compared to non-humidified rooms. There were small numbers of students ill with influenza like illnesses during our study so additional studies will need to look further at humidification as a way to reduce influenza infection and transmission.</jats:sec

Humidity as a non-pharmaceutical intervention for influenza A.

Influenza is a global problem infecting 5-10% of adults and 20-30% of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza virus distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza virus and its amount. Additionally samples were tested for their ability to infect cells in cultures. We observed a significant reduction (p < 0.05) in the total number of influenza A virus positive samples (air and fomite) and viral genome copies upon humidification as compared to control rooms. This suggests the future potential of artificial humidification as a possible strategy to control influenza outbreaks in temperate climates. There were 2.3 times as many ILI cases in the control rooms compared to the humidified rooms, and whether there is a causal relationship, and its direction between the number of cases and levels of influenza virus in the rooms is not known. Additional research is required, but this is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks

Influenza A virus genomic copies of positive samples.

Horizontal bars indicate mean copy number and error bars are 95% CI. Fomites control, n = 31; Fomites humidified, n = 27; Air (total) control, n = 33; Air (total) humidified, n = 21. * Indicates air samples calculated mean per cubic meter of air based on air sample volume. * P<0.001.</p

Influenza A positive samples by electrical impedance and RT-PCR assays from preschool.

Influenza A positive samples by electrical impedance and RT-PCR assays from preschool.</p