Identification Of Environmental,Viral And Host Factors Involved In The Transmission Of SARS-COV-2 And H1N1 Influenza Virus

1.BackgroundZoonotic respiratory viruses are important pathogens of recent emerging infectious diseases,such as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)causing the pandemic of coronavirus disease 2019(COVID-19)and avian influenza viruses causing influenza outbreaks.In addition to the virulence and pathogenicity of the virus itself,the transmissibility also has a great impact on public health.During the COVID-19 pandemic,later SARS-CoV-2 variants,such as Omicron,caused milder disease compared to earlier variants but transmitted more efficiently.Despite the milder disease caused by Omicron variants,their greater transmissibility led to a significant impact on public health.The highly pathogenic H5N1 avian influenza viruses that caused an outbreak in 2004 caused severe disease in humans with recorded mortality rates of up to 50%.However,these viruses did not show robust transmission between humans and as such the public health impact was less compared to the H7N9 viruses that caused outbreaks in 2013-2017.These H7N9 viruses were of low pathogenicity and caused a much milder disease compared to the H5N1 viruses,but could transmit more effectively between humans,leading to a greater impact on public health over a greater length of time.The transmission of respiratory viruses between humans and from animals to humans is dependent on multiple factors including the environment,the virus and the host.This is an important reason why transmission is not well understood,despite the fact that it is a critical part of the threat these viruses pose to public health.Avian influenza virus A/ruddy turnstone/Delaware/300/2009(H1N1)(DE300)can be airborne transmissible in ferrets.After mutating the amino acid at 213 of NS1 protein from serine(S)to proline(P),the airborne transmissibility was lost(DE213),indicating that this site is critical to the airborne transmissibility.Transcriptomic sequencing of turbinate tissues from DE300 and DE213-infected ferrets revealed significant differences in NLRP3(NOD-like receptor family,pyrin domain containing 3)expression levels,and NLRP3 inflammasome activation could promoting the maturation and secretion of cytokines IL-18(Interleukin-18)and IL-1β(Interleukin-1β)which is an important part of the host’s innate immune response,suggesting that NS1 may alter the transmissible phenotype of influenza virus by affecting the activation of the NLRP3 inflammasome,but the mechanism remains unknown.2.ObjectivesTo study environmental,viral,and host factors involved in the transmission of respiratory viruses SARS-CoV-2 and HIN1 influenza virus.Specifically:(1)to determine the transmissibility of SARS-CoV-2 in different healthcare settings by studying the environmental contamination proximal to COVID-19 patients;(2)to determine the critical sites of the influenza virus NS1 protein antagonizing host innate responses;(3)to explore the effect of the NLRP3 inflammasome activation on the pathogenicity and replication ability of influenza virus.3.Methods(1)We studied the environmental contamination associated with patients at different stages and severity of COVID-19 by collecting 400 environmental air and surface samples from the intensive care unit(ICU)and a general isolation ward of the First Affiliated Hospital of Guangzhou Medical University.The collection locations included general working areas used by medical staff and areas near patients.We detected the presence of SARS-CoV-2 RNA by quantitative PCR(qPCR)and correlated this dataset to the clinical and viral load data of the COVID-19 patients to correlate the environment to the patient.(2)The role of influenza virus NS1 as an antagonist of the host innate immune response was studied using an in vitro cell-based transfection assay whereby the effect of mutations in NS1,introduced by site-directed mutagenesis,on the expression of IL-1β,a cellular marker of the antiviral responses of cells.(3)Viral pathogenesis and replication were studied under pharmacological modulation of innate immune response in vitro using a cell-based infection assay.The cytotoxicity of drugs in different concentration ranges is tested at the same time.4.Results(1)Among the 218 ICU samples,we found one air sample collected from a patient’s bedside tested positive for SARS-CoV-2 RNA.Of the 182 specimens from general isolation wards,samples collected from face masks,floors,mobile phones,and the air in the wards and toilets tested positive for SARS-CoV-2 RNA.(2)Compared to the human-origin influenza viruses that show robust mammalian transmission,the NS1 of avian influenza viruses we studied here,which do not transmit to the same efficiency in mammalian models,significantly reduced the expression of IL-1β in vitro,which was indicative of differential regulation of the NLRP3 pathway.As we observed significant differences in the activities of NS1 between the human and avian viruses studied,we compared the amino acids that differed between the two groups and found that L7S(Leucine 7 Serine)and D171A(Aspartic acid 171 Alanine)in the avian influenza virus NS1,which changed these amino acids to those found in the human viruses,significantly reduced the inhibitory effect on IL-1β expression observed,whilst mutation of the other amino acids had little effect.We further analyzed the sequence of these two positions and found they are relatively conserved in avian and human,with S at the percentage of 55.3%,D of 53.7%and S at 98.4%,Y(Tyrosine)at 84.3%respectively.(3)Our studies of NLRP3 inhibitors revealed them to be non-toxic at concentrations below 100 μM in three types of mammalian cells,namely A549,MDCK and THP-1.Further,these drugs did not impact viral replication in these cells at non-toxic concentrations,indicating their potential to study activation of the NLRP3 pathway without altering peak viral titers.5.Conclusions(1)Compared with the general isolation ward,SARS-CoV-2 environmental contamination was much less in the ICU,even though patients in the isolation ward were recovered from severe COVID-19.These findings indicated that the infection control measures in the ICU were effective and that risks of nosocomial infections may be greater in the isolation ward.Therefore,care should be taken by hospital staff to prevent transmission of SARS-CoV-2 even in those patients that have recovered from severe COVID-19.(2)The effect of influenza virus NS1 on the NLRP3 pathway was significantly different between the avian and human origin viruses tested here,which was mediated by amino acids at the position of 7 and 171 of NS1.As the identities of these sites were relatively conserved within human and avian origin viruses,but differed between these groups,suggests that this may represent an important adaptation of zoonotic influenza viruses that facilitates mammalian transmissibility.(3)The NLRP3 inhibitor MCC950 can regulate the NLRP3 signaling pathway without producing cytotoxicity and affecting the virus replication ability,and can be used for the subsequent research on the relationship between the NLRP3 signaling pathway and virus transmission.