Hemagglutinin Cleavage Site Research Articles

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo

ABSTRACT To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus

Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.

Molecular Characterizations of a High Pathogenic Avian Influenza H5N8 in Iraq

High pathogenicity avian influenza subtype H5N8 viruses were detected in different areas in Iraq at thelast 2017 and early 2018 and 2019 and the disease was detected clinically. This disease is responsible forhigh economic losses for poultry industry and threat human health, so that, this study was conducted formolecular detection, characterization and phylogenic analysis of avian influenza in Iraq. AI subtype H5N8 isan infectious disease primarily in birds and responsible for severe respiratory illness which associated witha high percentage of morbidity and mortality in wild and domestic birds. During this study one hundred fiftydifferent samples including (trachea, larynx and lung) were collected from different areas of broiler chickenfrom Baghdad and its surrounding regions during winter 2019. Avian influenza virus subtype H5N8 wasdetected by using real time RT-PCR technique, and specific kits (KyltR Germany) for AIV subtypes H5 andN8 were used respectively. The results revealed that (15) samples out of (150) collected samples were gavepositive results for avian influenza H5 these positive samples were prepared for a second step of detectionby RT-PCR test specific for N8 subtype the results revealed that only (12) out of (15) tested samples werepositive for H5N8 (8%).Genetic sequencing of isolates and phylogenic analysis of three selected isolates of(H5N8) bellowing to different areas indicate that all strains bellowing to H5 class (2.3.4.4) high pathogenicavian influenza revealed that they are closely related to Egyptian strain (A/ duck/ Egypt/ F446/2017.H5N8-MH893737.1) (with 97.6% identity). Analysis of the mono-basic amino acid (PQIEPR / GLF) atthe hemagglutinin cleavage site revealed there is no deletion of the stalk region with the neuraminidaseindicated that the isolates is a typical HPAI strain (A / duck / Egypt / F446/2017). The similarity of thenucleotide sequence analysis of hemagglutinin gene revealed that there was a high homology (97.6%) to thatof A/duck/Egypt/F446/2017 H5N8 .

Molecular characterization of low pathogenic avian influenza H9N2 virus during co-circulation with newly-emerged highly pathogenic avian influenza H5N8 virus in Iran.

Identification of molecular characteristics of low pathogenic avian influenza (LPAI) H9N2 virus provides insights into the evolution of this subtype due to the modulation of genomic characteristics in co-circulation with another subtype. The present study aimed to analyze the molecular and phylogenetic characteristics of the current LPAI H9N2 virus in characteristics of internal proteins are crucial for the adaptations of AIVs viruses to a new host. Since H9N2 is indigenous among poultry, continuous monitoring of viral genetic changes is needed for risk assessment of potential transmissibility to human population and emergence of new reassortant virus. domestic poultry during the emergence of new highly pathogenic avian influenza (HPAI) H5N8 virus in Iran. To this end, deep sequencing of LPAI H9N2 virus was performed on Illumina MiSeq sequencing platform and the complete sequences of avian influenza viruses were obtained from GISAID EpiFlu database. Phylogenetic analysis of the surface and internal gene segments showed that the H9N2 2018 virus was closely related to Pakistani H9N2 isolates. HA cleavage site motif sequence of the Iranian isolate was 317KSSR GLF323. The A/chicken/Iran/1/2018 H9N2 strain carried the amino acid substitution (Q216L), which is a mutation correlated with a shift in the affinity of the HA from avian type sialic receptors to human type. Besides surface glycoproteins, molecular. Keywords: A/H9N2; molecular characterization; A/H5N8; co-circulation; A/H5N1; Illumina MiSeq.

A bivalent live attenuated influenza virus vaccine protects against H1N2 and H3N2 viral infection in swine

Swine Influenza A virus (swIAV) poses a substantial burden to the swine industry due to its highly contagious nature, acute viral disease, and ability to cause up to 100 % morbidity. Currently, North American swine are predominately infected with three subtypes of swIAV: H1N1, H1N2, and H3N2. The ability of influenza viruses to cross both directions between humans and swine means that both human and swine-origin viruses as well as new reassortant viruses can pose a substantial public health or pandemic threat. Since the primary method of protection and control against influenza is through vaccination, more effective, new vaccine platforms need to be developed. This study uses two Canadian swIAV isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69] to design a bivalent live attenuated influenza virus vaccine (LAIV) through reverse genetics. The hemagglutinin (HA) cleavage site from both SD191-WT and SD69-WT were engineered from a trypsin-sensitive to an elastase-sensitive motif, to generate SD191-R342V and SD69-K345V, respectively. The elastase dependent SD191-R342V virus possesses a mutation from arginine to valine at amino acid (aa) 342 on HA, whereas the elastase dependent SD69-K345V virus possesses a mutation from lysine to valine at aa 345 on HA. Both elastase dependent swIAVs are completely dependent on elastase, display comparable growth properties to the wild type (WT) viruses, are genetically stable in vitro, and entirely non-virulent in pigs. Moreover, when these elastase dependent swIAVs were administered together in pigs, they were found to stimulate antibody responses and IFN-γ secreting cells, as well as prevent viral replication and lung pathology associated with WT H1N2 and H3N2 swIAV challenge. Therefore, this bivalent LAIV demonstrates the strong candidacy to protect swine against the predominant influenza subtypes in North America.

Transcriptome Profiles of Highly Pathogenic Pure Avian H7N9 Virus-Infected Lungs of BALB/c Mice

Avian influenza A (H7N9) viruses emerged in China in 2013 and caused a zoonotic disease associated with a high case-fatality ratio of more than 30%. Transcriptional profiles obtained using animal models reveal host responses to the disease, thereby providing insights into disease pathogenesis. Therefore, we aimed to characterize the host responses of the H7N9 virus infected-mouse lungs in this study. First, we isolated an avian-originated H7N9 strain, which was shown to be highly pathogenic to both chickens and mice. Genomic analysis results suggested that a 12-nucleotide-insertion was present at the hemagglutinin cleavage site, and both the hemagglutinin and neuraminidase genes belonged to the Yangtze River Delta lineage. RNA sequencing results revealed 566 differentially expressed genes in the H7N9-infected lungs. Moreover, transcriptome analysis revealed that over-activated antiviral signals and intense interferon-stimulated gene products possibly contributed to the high virulence of the virus in mice. Importantly, lung concentrations of inflammatory cytokines, including interleukin-1β and interleukin-6, interferon-β, and tumor necrosis factor-α, were upregulated in response to H7N9 virus infection. Overall, the present study provided a comprehensive understanding of H7N9 virus pathogenicity and correlated host immune responses.

Molecular characteristics of H9N2 influenza viruses isolated from farmed raccoon dogs and arctic foxes in China

In this study, eight H9N2 IAVs were isolated from infected diseased, farmed raccoon dogs and arctic foxes. Eight genes shared 98.6%–100% identity among the isolates possessing a PSRSSR/GL motif at the HA cleavage site, which is same as the motif of G1 and Y280 lineages of H9N2 IAVs. The phylogenetic analysis showed that the HA genes of the eight isolates clustered with Y280-like viruses, whereas the NA genes belonged to F/98-like sublineage. Interestingly, the NS, NP, PB2 and PA genes of the isolates were closely related to H7N9 IAVs. This is the first evidence for isolation of H9N2 IAVs from raccoon dogs and arctic foxes. Raccoon dogs and arctic foxes potentially serve as an intermediate host for influenza viruses with pandemic potential toward other animals due to co-expression of both SA α-2,6-Gal and SA α-2,3-Gal receptors in a wide range of their tissues.

Phylogeographic analysis of H5N1 highly pathogenic avian influenza virus isolated in Cambodia from 2018 to 2019.

Since 2004, several outbreaks of highly pathogenic avian influenza (HPAI) have been reported in Cambodia. Until 2013, all H5N1 viruses identified in Cambodia belonged to clade 1 and its subclades. H5N1 HPAI viruses belonging to clade 2.3.2.1c have been dominant since the beginning of 2014, with various genotypes (KH1-KH5) reported. Here, we isolated nine H5N1 HPAI viruses from domestic poultry farms and slaughterhouses in Cambodia during 2018-2019 and performed phylogenetic analysis of whole genome sequences. All isolates were classified as H5 clade 2.3.2.1c viruses and all harbored multi-basic amino acid sequences (PQRERRRKR/GLF) at the haemagglutinin (HA) cleavage site. Phylogenetic analysis revealed that the H5N1 isolates in this study belonged to the KH2 genotype, the dominant genotype in Cambodia in 2015. Phylogenetic analysis of the HA gene showed that the isolates were divided into two groups (A and B). The results of Bayesian discrete phylogeography analysis revealed that the viral migration pathways from Vietnam to Cambodia (Bayes factor value: 734,039.01; posterior probability: 1.00) and from Cambodia to Vietnam (Bayes factor value: 26,199.95; posterior probability: 1.00) were supported by high statistical values. These well-supported viral migrations between Vietnam and Cambodia demonstrate that viral transmission continued in both directions. Several factors may have contributed to this, including the free-grazing duck system and movement of poultry-related products. Thus, the results emphasize the need for an enhanced international surveillance program to better understand transboundary infection and evolution of H5N1 HPAI viruses, along with implementation of more stringent international trade controls on poultry and poultry products.

Emergence of highly pathogenic H5N2 and H7N1 influenza A viruses from low pathogenic precursors by serial passage in ovo.

Highly pathogenic (HPAI) strains emerge from their low pathogenic (LPAI) precursors and cause severe disease in poultry with enormous economic losses, and zoonotic potential. Understanding the mechanisms involved in HPAI emergence is thus an important goal for risk assessments. In this study ostrich-origin H5N2 and H7N1 LPAI progenitor viruses were serially passaged seventeen times in 14-day old embryonated chicken eggs and Ion Torrent ultra-deep sequencing was used to monitor the incremental changes in the consensus genome sequences. Both virus strains increased in virulence with successive passages, but the H7N1 virus attained a virulent phenotype sooner. Mutations V63M, E228V and D272G in the HA protein, Q357K in the nucleoprotein (NP) and H155P in the neuraminidase protein correlated with the increased pathogenicity of the H5N2 virus; whereas R584H and L589I substitutions in the polymerase B2 protein, A146T and Q220E in HA plus D231N in the matrix 1 protein correlated with increased pathogenicity of the H7N1 virus in embryos. Enzymatic cleavage of HA protein is the critical virulence determinant, and HA cleavage site motifs containing multibasic amino acids were detected at the sub-consensus level. The motifs PQERRR/GLF and PQRERR/GLF were first detected in passages 11 and 15 respectively of the H5N2 virus, and in the H7N1 virus the motifs PELPKGKK/GLF and PELPKRR/GLF were detected as early as passage 7. Most significantly, a 13 nucleotide insert of unknown origin was identified at passage 6 of the H5N2 virus, and at passage 17 a 42 nucleotide insert derived from the influenza NP gene was identified. This is the first report of non-homologous recombination at the HA cleavage site in an H5 subtype virus. This study provides insights into how HPAI viruses emerge from low pathogenic precursors and demonstrated the pathogenic potential of H5N2 and H7N1 strains that have not yet been implicated in HPAI outbreaks.

Ser-Leu substitution at P2 position of the hemagglutinin cleavage site attenuates replication and pathogenicity of Eurasian avian-like H1N2 swine influenza viruses

Swine influenza viruses not only constitute a potential economic problem for livestock, but also pose a substantial threat to human health. Mutation in the proteolytic cleavage site of hemagglutinin (HA) is recognized as an essential factor of tissue tropism and viral pathogenicity. However, the molecular properties of the cleavage site of Eurasian avian-like swine (EA) H1N2 virus remain largely unknown. In this study, we found a serine-leucine (Ser-Leu) substitution at the P2 position of the HA cleavage site (S328 L) in naturally occurring EA H1N2 virus. To study the effect of this substitution, we used reverse genetics to generate recombinant wild-type and mutant viruses containing a single amino acid mutation at the P2 position in A/swine/Guangdong/YJ28/2014 (YJ28) or A/swine/Guangdong/DG2/2015 (DG2) background. In vitro experiments showed that the Ser-Leu substitution at the P2 position attenuated the viral replication and HA cleavage efficiency. In vivo analyses revealed that, while all mice inoculated with r/DG2-S328 L or r/YJ28 viruses survived, the survival rates of r/DG2- and r/YJ28-L328S-inoculated animals were 20 % and 40 %, respectively. Furthermore, the Ser-Leu substitution at the P2 position attenuated the replication in nasal turbinate and lungs. In summary, this amino acid change may be useful to understand the molecular properties of the cleavage site and be valuable for vaccine development.

Analysis on genetic characteristics of H9N2 avian influenza virus isolated from human infection and external environment in Gansu province

Objective: To analyze the genomic characteristics of human infection with H9N2 avian influenza virus in Gansu province. Methods: The etiological analysis was conducted for human infection with H9N2 avian influenza virus detected in influenza like illness cases in northwestern China in 2016. Molecular bioinformatics Mega 7.0 software was used to analyze the full genomic sequences of the viral isolate. Results: The gene fragments of HA, NA, MP, NP, NS, PA, PB1 and PB2 of the isolate were highly similar (>90%) to those of H9N2 avian influenza virus strain isolated in external environment in Gansu from 2014 to 2019. The HA gene belonged to BJ/94-like branch, PB2 and MP belonged to G1/97-like branch, and the PB1, PA, NS, and NP genes belonged to F/98-like branch. MP and PB2 were closely related to H7N9, H10N8 and H5N6 viruses. Amino acid sequence alignment showed that the HA cleavage site was arranged in PSRSSR ↓ GLF, H183N and Q226L mutated which included 7 HA glycosylated sites; 62-64 sites of NA absented 3 amino acids (ITE); and M2-31N, NS1-42S, PA-356R, and PA-409N mutated. Conclusions: Apparently, this case of human infection with human infection with H9N2 avian influenza virus was an incidental. However, the isolates of H9N2 influenza virus in external environment of Gansu had a series of mammalian adaptive molecular markers, suggesting that the risk of human infection is higher. It is necessary to strengthen the surveillance by multi departments to deal with influenza pandemic.

Genetic Characterization of a Novel North American-Origin Avian Influenza A (H6N5) Virus Isolated from Bean Goose of South Korea in 2018.

The complex overlap in waterfowl migratory pathways across the world has established numerous occurrences of genetic reassortment and intercontinental spread of avian influenza virus (AIV) over long distances, thereby calling for huge efforts and targeted surveillance for infection control. During annual surveillance in South Korea in 2018, a novel avian influenza H6N5 (K6) subtype was isolated from the fecal sample of wild bird. Genomic characterization using a phylogenetic tree indicated the K6 virus to be of North American-origin, with partial homology to an H6N5 strain, A/Aix galericulata/South Korea/K17-1638-5/2017 (K17). A monobasic residue at the HA cleavage site and absence of a notable mutation at the HA receptor-binding site suggested the isolate to be of low pathogenicity. However, molecular analysis revealed the E119V mutation in the NA gene and a human host marker mutation E382D in the polymerase acidic (PA) gene, implying their susceptibility to neuraminidase inhibitors and potential infectivity in humans, respectively. For comparison, K6 and K17 were found to be dissimilar for various mutations, such as A274T of PB2, S375N/T of PB1, or V105M of NP, each concerning the increased virulence of K6 in mammalian system. Moreover, kinetic data presented the highest viral titer of this H6N5 isolate at 106.37 log10TCID50 after 48 h of infection, thus proving efficient adaptability for replication in a mammalian system in vitro. The mouse virus challenge study showed insignificant influence on the total body weight, while viral load shedding in lungs peaked at 1.88 ± 0.21 log10 TICD50/mL, six days post infection. The intercontinental transmission of viruses from North America may continuously be present in Korea, thereby providing constant opportunities for virus reassortment with local resident AIVs; these results hint at the increased potential risk of host jumping capabilities of the new isolates. Our findings reinforce the demand for regular surveillance, not only in Korea but also along the flyways in Alaska.

Full-length genome sequences of the first H9N2 avian influenza viruses isolated in the Northeast of Algeria

BackgroundH9N2 avian influenza viruses (AIV) has a worldwide geographic distribution and affects poultry of different types of production. H9N2 AIV was first reported in the Northeast of Algeria in April 2017, following an outbreak associated with high mortality, in broiler flocks. In the present study, we report full-length genome sequences of AIV H9N2, and the detailed phylogeny and molecular genetic analyses.MethodsTen AIV H9N2 strains, collected in broiler flocks, were amplified in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Their full-length genomes were successfully sequenced and phylogenetic and molecular characterizations were conducted.ResultsPhylogenetic analysis showed that the isolates were monophyletic, grouped within the G-1 lineage and were very close to Moroccan and Algerian strains identified in 2016 and 2017, respectively. The low pathogenicity of the strains was confirmed by the sequence motif (335RSSR/GLF341) at the hemagglutinin (HA) cleavage site. An exclusive substitution (T197A) that had not been previously reported for H9N2 viruses; but, conserved in some pandemic H1N1 viruses, was observed. When compared to the G1-like H9N2 prototype, the studied strains showed one less glycosylation site in HA, but 2–3 additional ones in the stalk of the neuraminidase (NA). The HA protein harbored the substitution 234 L, suggesting binding preference to human-like receptors. The NA protein harbored S372A and R403W substitutions, previously detected in H9N2 from Asia and the Middle East, and especially in H2N2 and H3N2 strains that caused human pandemics. Different molecular markers associated with virulence and mammalian infections have been detected in the viral internal proteins. The matrix M2 protein possessed the S31N substitution associated with drug resistance. The non-structural 1 (NS1) protein showed the “GSEV” PDZ ligand (PL) C-terminal motif and no 80–84 deletion.ConclusionCharacterized Algerian AIV isolates showed mutations that suggest increased zoonotic potential. Additional studies in animal models are required to investigate the pathogenicity of these H9N2 AIV strains. Monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. Implementation of adequate biosecurity measures that limit the introduction and the propagation of AIV H9N2 in Algerian poultry farm is crucial.

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.Electronic supplementary materialThe online version of this article (10.1007/s11262-020-01764-2) contains supplementary material, which is available to authorized users.

A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice.

Avian influenza H7N9 viruses continue to pose a great threat to public health, which is evident by their high case-fatality rates. Although H7N9 was first isolated in humans in China in 2013, to date, there is no commercial vaccine available against this particular strain. Our previous studies developed a replication-defective influenza virus through mutation of the hemagglutinin (HA) cleavage site from a trypsin-sensitive to an elastase-sensitive motif. In this study, we report the development of a reassortant mutant influenza virus derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], which is the QVT virus. The HA gene of this virus possesses three mutations at the cleavage site, Lys-Gly-Arg were mutated to Gln-Thr-Val at amino acid (aa) positions 337, 338, and 339, respectively. We report this virus to rely on elastase in vitro, possess unaltered replication abilities when elastase was provided compared to the wild type virus in vitro, and to be non-virulent and replication-defective in mice. In addition, we report this virus to induce significant levels of antibodies and IFN-γ and IL-5 secreting cells, and to protect mice against a lethal challenge of the BC15 (H7N9) virus. This protection is demonstrated through the lack of body weight loss, 100% survival rate, and the prevention of BC15 (H7N9) viral replication as well as the reduction of proinflammatory cytokines induced in the mouse lung associated with the influenza disease. Therefore, these results provide strong evidence for the use of this reassortant mutant H7N9 virus as a replication-defective virus vaccine candidate against H7N9 viruses.

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)-Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Genetic Characterization of Avian Influenza A (H11N9) Virus Isolated from Mandarin Ducks in South Korea in 2018.

In July 2018, a novel avian influenza virus (A/Mandarin duck/South Korea/KNU18-12/2018(H11N9)) was isolated from Mandarin ducks in South Korea. Phylogenetic and molecular analyses were conducted to characterize the genetic origins of the H11N9 strain. Phylogenetic analysis indicated that eight gene segments of strain H11N9 belonged to the Eurasian lineages. Analysis of nucleotide sequence similarity of both the hemagglutinin (HA) and neuraminidase (NA) genes revealed the highest homology with A/duck/Kagoshima/KU57/2014 (H11N9), showing 97.70% and 98.00% nucleotide identities, respectively. Additionally, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds. Both the polymerase acidic (PA) and polymerase basic 1 (PB1) genes were close to the H5N3 strain isolated in China; whereas, other internal genes were closely related to that of avian influenza virus in Japan. A single basic amino acid at the HA cleavage site (PAIASR↓GLF), the lack of a five-amino acid deletion (residue 69–73) in the stalk region of the NA gene, and E627 in the polymerase basic 2 (PB2) gene indicated that the A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate was a typical low-pathogenicity avian influenza. In vitro viral replication of H11N9 showed a lower titer than H1N1 and higher than H9N2. In mice, H11N9 showed lower adaptation than H1N1. The novel A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate may have resulted from an unknown reassortment through the import of multiple wild birds in Japan and Korea in approximately 2016–2017, evolving to produce a different H11N9 compared to the previous H11N9 in Korea (2016). Further reassortment events of this virus occurred in PB1 and PA in China-derived strains. These results indicate that Japanese- and Chinese-derived avian influenza contributes to the genetic diversity of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) in Korea.

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico.

Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24-nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.

H7N9 Influenza Virus Containing a Polybasic HA Cleavage Site Requires Minimal Host Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.