Surveillance and Genetic Analysis of Low-Pathogenicity Avian Influenza Viruses Isolated from Feces of Wild Birds in Mongolia, 2021 to 2023.

The introduction of novel highly pathogenic (HPAI) viruses into Korea has been attributed to recombination events occurring at breeding sites in the Northern Hemisphere. This has increased interest in monitoring and genetically analyzing avian influenza viruses (AIVs) in northern regions, such as Mongolia, which share migratory bird flyways with Korea. AIVs in Mongolia were monitored by analyzing 10,149 fecal samples freshly collected from wild birds from April to October in 2021 to 2023. The prevalence of AIVs in wild birds was 1.01%, with a total of 77 AIVs isolated during these 3 years. These 77 AIVs included hemagglutinin (HA) subtypes H1, H2, H3, H4, H6, H10 and H13 and neuraminidase (NA) subtypes N1, N2, N3, N6, N7 and N8. The most frequently detected subtype combinations were H3N8 (39.0%) and H4N6 (19.5%), although HPAI viruses were not detected. Genetic analysis indicated that theses AIVs isolated from Mongolian samples were closely related to AIVs in wild birds in Korea, including those of Eurasian lineage. These findings indicate the necessity of continuous AIV surveillance and monitoring, as HPAI viruses introduced into Korea may derive from strains in Mongolia.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNß, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

A longitudinal molecular and cellular lung atlas of lethal SARS-CoV-2 infection in K18-hACE2 transgenic mice.

BACKGROUND: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 500 million cases and 6 million deaths worldwide. Previous investigations into the pathophysiology of SARS-CoV-2 primarily focused on peripheral blood mononuclear cells from patients, lacking detailed mechanistic insights into the virus's impact on inflamed tissue. Existing animal models, such as hamster and ferret, do not faithfully replicate the severe SARS-CoV-2 infection seen in patients, underscoring the need for more relevant animal system-based research. METHODS: In this study, we employed single-cell RNA sequencing (scRNA-seq) with lung tissues from K18-hACE2 transgenic (TG) mice during SARS-CoV-2 infection. This approach allowed for a comprehensive examination of the molecular and cellular responses to the virus in lung tissue. FINDINGS: Upon SARS-CoV-2 infection, K18-hACE2 TG mice exhibited severe lung pathologies, including acute pneumonia, alveolar collapse, and immune cell infiltration. Through scRNA-seq, we identified 36 different types of cells dynamically orchestrating SARS-CoV-2-induced pathologies. Notably, SPP1+ macrophages in the myeloid compartment emerged as key drivers of severe lung inflammation and fibrosis in K18-hACE2 TG mice. Dynamic receptor-ligand interactions, involving various cell types such as immunological and bronchial cells, defined an enhanced TGFß signaling pathway linked to delayed tissue regeneration, severe lung injury, and fibrotic processes. INTERPRETATION: Our study provides a comprehensive understanding of SARS-CoV-2 pathogenesis in lung tissue, surpassing previous limitations in investigating inflamed tissues. The identified SPP1+ macrophages and the dysregulated TGFß signaling pathway offer potential targets for therapeutic intervention. Insights from this research may contribute to the development of innovative diagnostics and therapies for COVID-19. FUNDING: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020M3A9I2109027, 2021R1A2C2004501).

SARS-CoV-2 infection engenders heterogeneous ribonucleoprotein interactions to impede translation elongation in the lungs.

Translational regulation in tissue environments during in vivo viral pathogenesis has rarely been studied due to the lack of translatomes from virus-infected tissues, although a series of translatome studies using in vitro cultured cells with viral infection have been reported. In this study, we exploited tissue-optimized ribosome profiling (Ribo-seq) and severe-COVID-19 model mice to establish the first temporal translation profiles of virus and host genes in the lungs during SARS-CoV-2 pathogenesis. Our datasets revealed not only previously unknown targets of translation regulation in infected tissues but also hitherto unreported molecular signatures that contribute to tissue pathology after SARS-CoV-2 infection. Specifically, we observed gradual increases in pseudoribosomal ribonucleoprotein (RNP) interactions that partially overlapped the trails of ribosomes, being likely involved in impeding translation elongation. Contemporaneously developed ribosome heterogeneity with predominantly dysregulated 5 S rRNP association supported the malfunction of elongating ribosomes. Analyses of canonical Ribo-seq reads (ribosome footprints) highlighted two obstructive characteristics to host gene expression: ribosome stalling on codons within transmembrane domain-coding regions and compromised translation of immunity- and metabolism-related genes with upregulated transcription. Our findings collectively demonstrate that the abrogation of translation integrity may be one of the most critical factors contributing to pathogenesis after SARS-CoV-2 infection of tissues.

Development of a Novel Korean H9-Specific rRT-PCR Assay and Its Application for Avian Influenza Virus Surveillance in Korea.

Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the "traditional" method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently.

Receptor binding motif surrounding sites in the spike 1 protein of infectious bronchitis virus have high susceptibility to mutation related to selective pressure.

BACKGROUND: To date, various genotypes of infectious bronchitis virus (IBV) have co-circulated and in Korea, GI-15 and GI-19 lineages were prevailing. The spike protein, particularly S1 subunit, is responsible for receptor binding, contains hypervariable regions and is also responsible for the emerging of novel variants. OBJECTIVE: This study aims to investigate the putative major amino acid substitutions for the variants in GI-19. METHODS: The S1 sequence data of IBV isolated from 1986 to 2021 in Korea (n = 188) were analyzed. Sequence alignments were carried out using Multiple alignment using Fast Fourier Transform of Geneious prime. The phylogenetic tree was generated using MEGA-11 (ver. 11.0.10) and Bayesian analysis was performed by BEAST v1.10.4. Selective pressure was analyzed via online server Datamonkey. Highlights and visualization of putative critical amino acid were conducted by using PyMol software (version 2.3). RESULTS: Most (93.5%) belonged to the GI-19 lineage in Korea, and the GI-19 lineage was further divided into seven subgroups: KM91-like (Clade A and B), K40/09-like, QX-like (I-IV). Positive selection was identified at nine and six residues in S1 for KM91-like and QX-like IBVs, respectively. In addition, several positive selection sites of S1-NTD were indicated to have mutations at common locations even when new clades were generated. They were all located on the lateral surface of the quaternary structure of the S1 subunits in close proximity to the receptor-binding motif (RBM), putative RBM motif and neutralizing antigenic sites in S1. CONCLUSIONS: Our results suggest RBM surrounding sites in the S1 subunit of IBV are highly susceptible to mutation by selective pressure during evolution.

Subtype specific virus enrichment with immunomagnetic separation method followed by NGS unravels the mixture of H5 and H9 avian influenza virus.

Wild bird avian influenza type A virus (AIV) surveillance is important for the early detection of highly pathogenic AIVs and for providing early warnings to the poultry industry and veterinary services to implement more effective control measures against these viruses. Some field samples are often found to contain more than two kinds of AIV. Correct determination of the HA/NA subtype and complete nucleotide sequences of the component viruses in those samples are often critical for timely and accurate understanding of the field situation, but it is not easy to define the genomic structure of the constituent viruses unambiguously because AIV has eight segmented genomes. In this study, with immunomagnetic beads incorporating polyclonal antibodies of chicken for subtype-specific viral enrichment, we could selectively decrease the density of one of the two constituent viruses in a sample of different subtypes, H5 and H9, artificially generated; this was represented in the changes of Ct values with subtype specific real-time RT-PCR. Following this, with NGS, we could recover nearly complete genomic sequences and arrange the consensus sequences of gene segments of the constituent viruses confidently with the quantitative variable like genome coverage, linked along the gene segments and associated with the number of viral copies in a sample.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

Statistical Analysis of the Performance of Local Veterinary Laboratories in Molecular Detection (rRT-PCR) of Avian Influenza Virus via National Proficiency Testing Performed during 2020-2022.

For the early detection of avian influenza virus (AIV), molecular diagnostic methods such as real-time RT-PCR (rRT-PCR) are the first choice in terms of accuracy and speed in many countries. A laboratory's capability to perform this diagnostic method needs to be measured through external and independent assessment to ensure that the method is validated within the laboratory and in interlaboratory comparison. The Animal and Plant Quarantine Agency of Korea has implemented five rounds of proficiency testing (PT) for rRT-PCR targeting local veterinary service laboratories involved in the AIV national surveillance program from 2020 to 2022. In each round, a portion composed of six or more samples was selected from the entire PT panel consisting of H5, H7, and H9 viruses isolated in Korea and distributed to each participant, and at least one pair of samples was commonly included in each panel for interlaboratory comparison. During the five rounds of PT, a few incorrect and outlying results were detected that required immediate inspection or corrective actions. However, in the quantitative measurement of Ct values, the average standard deviation or coefficient of variation decreased as multiple PT rounds progressed, and a positive correlation between consecutive rounds of PT was observed since 2021. The better consistency or stability in the experimental performance appeared to contribute to the more harmonized results in the latest PTs, and it is assumed that the positive reaction of participants to the challenges of quantitative assessment reports showing their status intuitively might work. We need to continue operating the PT program for local laboratories because they play crucial roles at the front line of the national avian influenza surveillance program, and frequent changes in the human resources or environment for diagnosis in those laboratories are inevitable.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Corrigendum: Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

[This corrects the article DOI: 10.3389/fimmu.2022.1055811.].

Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has been a global health concern since 2019. The viral spike protein infects the host by binding to angiotensin-converting enzyme 2 (ACE2) expressed on the cell surface, which is then processed by type II transmembrane serine protease. However, ACE2 does not react to SARS-CoV-2 in inbred wild-type mice, which poses a challenge for preclinical research with animal models, necessitating a human ACE2 (hACE2)-expressing transgenic mouse model. Cytokeratin 18 (K18) promoter-derived hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] are widely used for research on SARS-CoV-1, MERS-CoV, and SARS-CoV-2. However, SARS-CoV-2 infection is lethal at ≥105 PFU and SARS-CoV-2 target cells are limited to type-1 alveolar pneumocytes in K18-hACE2 mice, making this model incompatible with infections in the human lung. Hence, we developed lung-specific SARS-CoV-2 infection mouse models with surfactant protein B (SFTPB) and secretoglobin family 1a member 1 (Scgb1a1) promoters. After inoculation of 105 PFU of SARS-CoV-2 to the K18-hACE2, SFTPB-hACE2, and SCGB1A1-hACE2 models, the peak viral titer was detected at 2 days post-infection and then gradually decreased. In K18-hACE2 mice, the body temperature decreased by approximately 10°C, body weight decreased by over 20%, and the survival rate was reduced. However, SFTPB-hACE2 and SCGB1A1-hACE2 mice showed minimal clinical signs after infection. The virus targeted type I pneumocytes in K18-hACE2 mice; type II pneumocytes in SFTPB-hACE2 mice; and club, goblet, and ciliated cells in SCGB1A1-hACE2 mice. A time-dependent increase in severe lung lesions was detected in K18-hACE2 mice, whereas mild lesions developed in SFTPB-hACE2 and SCGB1A1-hACE2 mice. Spleen, small intestine, and brain lesions developed in K18-hACE2 mice but not in SFTPB-hACE2 and SCGB1A1-hACE2 mice. These newly developed SFTPB-hACE2 and SCGB1A1-hACE2 mice should prove useful to expand research on hACE2-mediated respiratory viruses.

Temporal Transcriptome Analysis of SARS-CoV-2-Infected Lung and Spleen in Human ACE2-Transgenic Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and potentially fatal virus. So far, most comprehensive analyses encompassing clinical and transcriptional manifestation have concentrated on the lungs. Here, we confirmed evident signs of viral infection in the lungs and spleen of SARS-CoV-2-infected K18-hACE2 mice, which replicate the phenotype and infection symptoms in hospitalized humans. Seven days post viral detection in organs, infected mice showed decreased vital signs, leading to death. Bronchopneumonia due to infiltration of leukocytes in the lungs and reduction in the spleen lymphocyte region were observed. Transcriptome profiling implicated the meticulous regulation of distress and recovery from cytokine-mediated immunity by distinct immune cell types in a time-dependent manner. In lungs, the chemokine-driven response to viral invasion was highly elevated at 2 days post infection (dpi). In late infection, diseased lungs, post the innate immune process, showed recovery signs. The spleen established an even more immediate line of defense than the lungs, and the cytokine expression profile dropped at 7 dpi. At 5 dpi, spleen samples diverged into two distinct groups with different transcriptome profile and pathophysiology. Inhibition of consecutive host cell viral entry and massive immunoglobulin production and proteolysis inhibition seemed that one group endeavored to survive, while the other group struggled with developmental regeneration against consistent viral intrusion through the replication cycle. Our results may contribute to improved understanding of the longitudinal response to viral infection and development of potential therapeutics for hospitalized patients affected by SARS-CoV-2.

Comparison of the pathogenesis of SARS-CoV-2 infection in K18-hACE2 mouse and Syrian golden hamster models.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extrapulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using preclinical models.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety.

For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3'-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.

Rank orders of mammalian pathogenicity-related PB2 mutations of avian influenza A viruses.

The PB2 gene is one of the key determinants for the mammalian adaptation of avian influenza A viruses (IAVs). Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different genetic backgrounds of avian IAVs, the relative effects of single or multiple mutations on viral fitness could not be directly compared. Furthermore, their mutational steps during mammalian adaptation had been unclear. In this study, we collectively compared the effects of individual and combined MPMs on viral fitness and determined their rank orders using a prototypic PB2 gene. Early acquired mutations may determine the function and potency of subsequent mutations and be important for recruiting multiple, competent combinations of MPMs. Higher mammalian pathogenicity was acquired with the greater accumulation of MPMs. Thus, the rank orders and the prototypic PB2 gene may be useful for predicting the present and future risks of PB2 genes of avian and mammalian IAVs.

Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin.

Clade 2.3.4.4c H5N6 avian influenza A viruses (AIVs) may have originally adapted to infect chickens and have caused highly pathogenic avian influenza (HPAI) in poultry and human fatalities. Although A/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant clade 2.3.4.4c H5N6 vaccine strains have been effective in embryonated chicken eggs-based vaccine production system, they need to be improved in terms of immunogenicity and potential mammalian pathogenicity. We replaced the PB2 gene alone or the PB2 (polymerase basic protein 2), NP (nucleoprotein), M (matrix protein) and NS (non-structural protein) genes together in the PR8 strain with corresponding genes from AIVs with low pathogenicity to remove mammalian pathogenicity and to match CD8+ T cell epitopes with contemporary HPAI viruses, respectively, without loss of viral fitness. Additionally, we tested the effect of the H103Y mutation of hemagglutinin (HA) on antigen productivity, mammalian pathogenicity and heat/acid stability. The replacement of PB2 genes and the H103Y mutation reduced the mammalian pathogenicity but increased the antigen productivity of the recombinant vaccine strains. The H103Y mutation increased heat stability but unexpectedly decreased acid stability, probably resulting in increased activation pH for HA. Interestingly, vaccination with inactivated recombinant virus with replaced NP, M and NS genes halted challenge virus shedding earlier than the recombinant vaccine without internal genes replacement. In conclusion, we successfully generated recombinant clade 2.3.4.4c H5N6 vaccine strains that were less pathogenic to mammals and more productive and heat stable than conventional PR8-derived recombinant strains by optimization of internal genes and the H103Y mutation of HA.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity.

Abstract: Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.