Mammalian Pathogenicity Study Of An H9N2 Influenza A Virus Isolated From A Severely Ill Nine-Year-Old Patient

BackgroundSubtype H9N2 avian influenza viruses(AIVs)pose significant risks to human and animal health,as they frequently reassort with other subtypes,‘donating’ their internal genes and giving rise to new viruses with increased zoonotic potential,such as the subtype H5N1 highly pathogenic AIVs isolated in 1997 and the subtype H7N9 influenza viruses that emerged in China in 2013.H9N2 AIVs cause mild disease in otherwise healthy individuals,with severe illness only associated with the very young or those with underlying medical conditions.Human cases are also relatively rare,with only 70 reported worldwide to date,the majoriy in China.Here we report a severe,non-fatal human case of influenza-like illness in an otherwise healthy nine-year-old in Suzhou,China associated with a H9N2 AIV highly similar to viruses isolated in poultry in the region.To our knowledge this is the first report of such a case in a healthy child.As such,we conducted a risk assessment on this virus,A/Suzhou/GIRD01/2019(H9N2)(SZ/GIRD01)to characterize the zoonotic potential of this virus in comparison to a contemporary H9N2 AIV isolated in poultry in the region,A/chicken/Zhejiang/198/2019(H9N2)(ZJ/198),and older prototypical H9N2 AIVs.ObjectiveThis project researched the mammalian pathogenicity and zoonotic potential of the human-isolated strain SZ/GIRD01 through genetic analysis and in vivo and in vitro experiments.Methods1.Clinical analysis: i.Analyze the characteristics of patients infected with H9N2 based on this patient records and other H9N2 infections reported in the literature;ii.Use Real Time PCR to detect viral in samples to evaluate viral spreading characteristics;iii.Analyze the patient epidemiological history determine the possible interfamily transmission events;iv.Perform second-generation sequencing to study viral evolution.2.viral isolation and culture: i.Isolate virus from patient;ii.Rescue viruses as control group by reverse genetics;iii.Grow viruses in chick embryo or MDCK cells;iv.Concertrate virus by ultracentrifugation in sucrose solution.3.Genetic analysis: i.Sequence SZ/GIRD01 strain;ii.Perform phylogenetic analysis to determine genotype and trance the origination of the SZ/GIRD01;iii.Identify the gene marker and analysis the genetic characteristics of SZ/GIRD01 strain by multiple sequence alignment.4.In vitro experiments: i.Growth kinetics were determined in MDCK cells;ii.Binding to different sialic acid analogs was measured by ELISA;iii.Neuraminidase activity was measured by MUNANA assay and ELLA assay;iv.Virus elution from RBCs was measured at different temperatures to study the binding affinity and sialidase activity;v.Determine viral drug susceptibility to neuraminidase inhbitors.5.Mouse model: Study the mammalian pathogenicity of these viruses by measuring weight loss.Serum was collected on the 28 th day after the infection and the production of serum antibodies was determined by the hemagglutination inhibition test.Results1.The patient presented with fever,vomiting but rapidly declined to progressive wheezing followed by dyspnea.He was admitted to the intensive care unit and placed on mechanical ventilation.A diagnosis of pneumonia and type I respiratory failure was made.Respiratory syncytial virus and influenza A viruses was also detected in bronchiolavelolar lavage.Following an aggressive regimen of antiviral and antibacterial therapy,the patient recovered and was discharged from hospital after 13 days.2.RNA of H9N2 was detected in the bronchiolavelolar lavage and anal swab specimens,suggesting lower lung and extrapulmonary involvement.3.The G57 genotype is currently the dominant genotype in China,and SZ/GIRD01 belongs to G57 genotype;4.Domestic H7N9,H9N2 and some H5 subtype strains share same internal genes,and the PA gene of SZ/GIRD01 strain cluster with a group of H7N9 s.Other 7gene segments are similar to some H9N2 viruses which were isolated in Yangtze river delta;5.G57 subtype strains carry some mammalian adaptive mutations,including HA-Q226 L,NA 62-64 amino acid deletion,PB2-E627 V,HA-T190 A,PA-K356 R.SZ/GIRD01 strain also carried HA-N158 D and PB2-V598 I,which;6.SZ/GIRD01 exhibits greater replication ability in mammalian cells compared to seasonal influenza strains(H1N1,H3N2)and old H9N2 strains(G1 and BJ94H9N2 lineage);7.SZ/GIRD01 bound to ɑ2,3 and ɑ2,6 sialic acids but showed a strong preference for human-type ɑ2,6 SLN’ sialic acids;8.SZ/GIRD01 was sensitive to neuraminidase inhibitors;9.SZ/GIRD01 strain can infect mice without pre-adaptation,causing mild infections with no symptomology.ConclusionOur virology data showed that SZ/GIRD01 was less pathogenic compared to ZJ198 but both viruses showed mammalian receptor binding preferences and an enhanced replication kinetics in MDCK cells compared to older H9N2 viruses.While SZ/GIRD01 was not more virulent than ZJ198,it displayed a binding preference for mammalian-type sialic acids and robust replication in mammalian cells.Phylogenetic analysis confirmed the epidemicity of G57 genotype viruses and extensive reassortment between inter-or intra-subtype viruses in China.And the genetic analysis indentified some mammalian pathogenicity marker in G57 genotype viruses.All of this indicated that currently circulating H9N2 AIVs pose a significant risk of zoonoses to humans.