Targeting influenza A virus by splicing inhibitor herboxidiene reveals the importance of subtype-specific signatures around splice sites.

BACKGROUND: The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene. METHODS: We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance. RESULTS: M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels. CONCLUSIONS: The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target.

Overview of Neutralization Assays and International Standard for Detecting SARS-CoV-2 Neutralizing Antibody.

We aimed to review the existing literature on the different types of neutralization assays and international standards for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We comprehensively summarized the serological assays for detecting neutralizing antibodies against SARS-CoV-2 and demonstrated the importance of an international standard for calibrating the measurement of neutralizing antibodies. Following the coronavirus disease outbreak in December 2019, there was an urgent demand to detect neutralizing antibodies in patients or vaccinated people to monitor disease outcomes and determine vaccine efficacy. Therefore, many approaches were developed to detect neutralizing antibodies against SARS-CoV-2, such as microneutralization assay, SARS-CoV-2 pseudotype virus assay, enzyme-linked immunosorbent assay (ELISA), and rapid lateral flow assay. Given the many types of serological assays for quantifying the neutralizing antibody titer, the comparison of different assay results is a challenge. In 2020, the World Health Organization proposed the first international standard as a common unit to define neutralizing antibody titer and antibody responses against SARS-CoV-2. These standards are useful for comparing the results of different assays and laboratories.

Stability of SARS-CoV-2 Spike G614 Variant Surpasses That of the D614 Variant after Cold Storage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (-20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at -20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at -20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant.IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at -20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and -20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.

Circulating pattern and genomic characteristics of influenza B viruses in Taiwan from 2003 to 2014.

BACKGROUND/PURPOSE: Influenza B viruses are antigenically classified into Yamagata and Victoria lineages according to their hemagglutinin (HA) proteins. These two lineages are known to either appear sequentially or cocirculate in Taiwan. METHODS: Taiwanese influenza B viral HA and neuraminidase (NA) sequences between 2003 and 2014 were determined and analyzed. A time-scaled phylogenetic tree was constructed to decipher the evolutionary trends of these sequences, and the reassortment between the two lineages. Positively selected amino acids were predicted, demonstrating the adaptive mutations of the circulating pattern. RESULTS: The HA phylogenetic tree revealed that the Victoria lineage evolved into a ladder-like pattern, whereas the Yamagata lineage exhibited complex topology with several independently evolved clades on which viruses from different influenza seasons interlaced. For several seasons, HA sequences were found to be dominated by strains of the same lineage as the corresponding vaccine strain. Inspecting these sequences revealed that frequent mutations occurred in neutralizing epitopes and glycosylation sites. Amino acid positions 212 and 214 of N-glycosylation sites, which are known to be critical determinants of receptor-binding specificity, were found to be subject to positive selection. No drug-resistant sites were noticed in the NA sequences. In addition, we identified several cases of NA reassortment with an overall incidence rate of 6% for the investigated Taiwan strains. CONCLUSION: We highlighted the interplay between mutations in the glycosylation sites and epitope during HA evolution. These are crucial molecular signatures to be monitored for influenza B epidemics in the future.