Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Zoonotic Threat of G4 Genotype Eurasian Avian-Like Swine Influenza A(H1N1) Viruses, China, 2020.

We investigated genetic and biologic characteristics of 2 Eurasian avian-like H1N1 swine influenza viruses from pigs in China that belong to the predominant G4 genotype. One swine isolate exhibited strikingly great homology to contemporaneous human Eurasian avian-like H1N1 isolates, preferential binding to the human-type receptor, and vigorous replication in mice without adaptation.

Effects of HA2 154 deglycosylation and NA V202I mutation on biological property of H5N6 subtype avian influenza virus.

In recent years, clade 2.3.4.4 H5N6 subtype avian influenza virus (AIV) has been predominantly prevalent in poultry flocks in China. During our AIV surveillance in 2018-2019, 6 circulating strains of H5N6 that possess the natural loss of glycosylation site 154 due to N154D mutation in HA2 protein were isolated. In particular, 5 strains simultaneously carried the V202I mutation in NA protein. Based on the paired backbone H5N6 viruses Y6 and RY6, which just diverged in the glycosylation status at site 158 in HA1 protein, 8 reassortants of rY6-154 N/202 V, rY6-154D/202 V, rY6-154 N/202I and rY6-154D/202I plus rRY6-154 N/202 V, rRY6-154D/202 V, rRY6-154 N/202I and rRY6-154D/202I were constructed with different variation patterns at site 154 in HA2 and site 202 in NA. By determining those reassortants in growth performance on cells, plaque-forming ability, heat and low pH stability, and pathogenicity in mammals, the results showed that HA2 N154D and NA V202I could singly or jointly change the viral biological properties both in vitro and in vivo. Additionally, the effect of HA mutation was significantly more robust than that of NA, and the resulting increasing or reducing impact was closely related to the glycosylation at HA1 site 158. The present study provided a reference for further parsing the relevant mechanism of the functional match between HA and NA proteins of the influenza virus.

Phylogenetic and phenotypic characterization of two novel clade 2.3.2.1 H5N2 subtype avian influenza viruses from chickens in China.

The extended co-circulation of H5 subtype highly pathogenic avian influenza (HPAI) viruses and H9N2 low pathogenic avian influenza (LPAI) viruses has inevitably facilitated gene reassortment between the two subtypes in fields. And, novel reassortant H5NX viruses harboring partial or even whole sets of H9N2 internal genes have continuously been detected, such as clade 2.3.4.4 H5N2 or H5N6 reassortants. Here, we report two novel H5N2 subtype HPAI isolates of HF9 and QY5 from chickens in live poultry markets during routine surveillance in 2018. Phylogenetic analysis showed that those two H5N2 strains both possessed the HA genes from clade 2.3.2.1e of H5N1 viruses but all the other seven gene segments consistently from the endemic S genotype of H9N2 subtype viruses. Further analysis revealed that HF9 and QY5 differed only in six sites including K353R, A588T and T661I in PB2, I682V and L704S in PB1 plus G631S in PA at the amino acid level. A chicken regression experiment confirmed that both HF9 and QY5 were lethal infection to all tested chickens via contact transmission. Moreover, those two isolates could immediately replicate in mice lungs without adaptation. However, mortality rate of those two variants were distinct in mice model, HF9 with 100% but QY5 with just 20% at the infection dosage of 106.0EID50 per mouse. We suppose that the phenotypic difference may probably be attributed to the amino acid substitutions in the polymerase genes between the two isolates that constitute of a subject of further ongoing research.

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11 years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

Genetic and antigenic diversity of H7N9 highly pathogenic avian influenza virus in China.

Avian influenza virus (AIV) H7N9 that emerged in 2013 in eastern China is a novel zoonotic agent mainly circulating in poultry without clinical signs but causing severe disease with high fatality in humans in more than 5 waves. Since the emergence of highly pathogenic (HP) H7N9 variants in 2016, it has induced heavy losses in the poultry industry leading to the implementation of an intensive nationwide vaccination program at the end of wave 5 (September 2017). To characterize the ongoing evolution of H7N9 AIV, we conducted analyses of H7N9 glycoprotein genes obtained from 2013 to 2019. Bayesian analyses revealed a decreasing population size of HP H7N9 variants post wave 5. Phylogenetic topologies revealed that two novel small subclades were formed and carried several fixed amino acid mutations that were along HA and NA phylogenetic trees since wave 5. Some of the mutations were located at antigenic sites or receptor binding sites. The antigenic analysis may reveal a significant antigenic drift evaluated by hemagglutinin inhibition (HI) assay and the antigenicity of H7N9 AIV might evolute in large leaps in wave 7. Molecular simulations found that the mutations (V135T, S145P, and L226Q) around the HA receptor pocket increased the affinity to α2,3-linked sialic acid (SIA) while decreased to α2,6-linked SIA. Altered affinity may suggest that HP H7N9 variations aggravate the pathogenicity to poultry but lessen the threat to public health. Selection analyses showed that the HP H7N9 AIV experienced an increasing selection pressure since wave 5, and the national implementation of vaccination might intensify the role of natural selection during the evolution waves 6 and 7. In summary, our data provide important insights about the genetic and antigenic diversity of circulating HP H7N9 viruses from 2017 to 2019. Enhanced surveillance is urgently warranted to understand the current situation of HP H7N9 AIV.

The Packaging Regions of G1-Like PB2 Gene Contribute to Improving the Survival Advantage of Genotype S H9N2 Virus in China.

The genotype S (G57) H9N2 virus, which first emerged in 2007 with the substitution of the G1-like PB2 gene for F98-like ones, has become the predominant genotype in the past 10 years. However, whether this substitution plays a role in the fitness of genotype S H9N2 viruses remains unknown. Comparison of the PB2 genes of F98-like and G1-like viruses revealed a close homology in amino acid sequences but great variations at nucleotide levels. We then determined if the packaging region, a unique sequence in each segment utilized for the assembly of the vRNA into virions, played a role in the fitness of the S genotype. The chimeric H9N2 virus with PB2 segments of the G1-like packaging regions significantly increased viral protein levels and polymerase activity. Substituting the packaging regions in the two terminals of F98-like PB2 with the sequence of G1-like further improved its competitive advantage. Substitution of the packaging regions of F98-like PB2 with those of G1-like sequences increased the infectivity of the chimeric virus in the lungs and brains of chicken at 3 days post infection (dpi) and extended the lengths of virus shedding time. Our study suggests that the packaging regions of the G1-like PB2 gene contribute to improve the survival advantage of the genotype S H9N2 virus in China.

Phylogenetic tracing and biological characterization of a novel clade 2.3.2.1 reassortant of H5N6 subtype avian influenza virus in China.

In recent years in China, clade 2.3.4.4 H5N6 plus clade 2.3.2.1 H5N1 subtype highly pathogenic avian influenza (HPAI) viruses have gradually become endemic in poultry, and their co-circulation could inevitably facilitate the gene reassortment between each other. During our routine surveillance in live poultry markets (LPMs) in eastern China in 2017-2018, a novel reassortant H5N6 strain with the HA gene derived from clade 2.3.2.1 was isolated from the cloacal swabs of apparently healthy ducks. Phylogenetic tracing analysis indicated that another two clade 2.3.2.1 H5N1 strains with divergent lineages of PB1 gene and one clade 2.3.4.4 H5N6 isolate of the dominant genotype sharing spatio-temporal proximity were intimately involved in the generation of this rarely reported clade 2.3.2.1 H5N6 reassortant. Distinct with the other three HPAI H5 viruses showing moderate virulence in mice, the H5N1 strain of the homologous internal gene constellation against the clade 2.3.2.1 H5N6 reassortant was highly pathogenic, which might probably attribute to the H3 subtype-derived PB1 gene. However, as compared to the clade 2.3.4.4 H5N6 ancestor, the clade 2.3.2.1 H5N6 reassortant displayed a broader tissue distribution and higher viral titres in mice, which could likely facilitate the viral maintenance and spread in nature. Therefore, our results highlight that continuous epidemiological survey of H5 subtype HPAI viruses in LPMs needs to be strengthened to prevent the potential poultry or even public health threat of the novel reassortants from endemic viruses.

Novel Reassortant H3N2 Avian Influenza Virus Isolated from Domestic Ducks in Eastern China in 2016.

H3 subtype avian influenza virus (AIV) poses a great threat to public health, and so investigating its epidemiology is of great importance. A novel reassortant H3N2 AIV strain was isolated from a live poultry market in eastern China. The strain's genes originated from H1N1, H3, and H7 AIVs. Thus, the genome information of the H3N2 isolate will help to investigate further the epidemiology of H3 subtype AIVs in China.

CuCl/CCl(4)-promoted convenient synthesis of sulfonyl amidines from tertiary amines and sulfonyl azides.

Promoted by CuCl/CCl(4), a variety of sulfonyl azides and tertiary amines were successfully coupled to give sulfonyl amidine derivatives in good to excellent yields. A possible mechanism for this reaction is discussed.