Microparticles from human the lower airway show inhibitory activity against respiratory syncytial virus.

Airway microparticles (MPs) have been shown previously to inhibit influenza virus by trapping virions on their surface through their surface viral receptor. It was hypothesized that airway MPs may carry most of the epithelial cell surface molecules, including receptors for respiratory viruses, and may be able to inhibit various respiratory viruses. We show here that MPs from human bronchoalveolar lavage (BAL) can inhibit respiratory syncytial virus (RSV). Those MPs stained positive for the RSV receptor, CX3CR1. Furthermore, incubating the MPs with a monoclonal antibody against CX3CR1 reduced the anti-RSV activity. These data indicate that MPs can contribute to respiratory innate antiviral defense.

Responses to the Sb epitope contributed to antigenic drift of the influenza A 2009 H1N1 virus.

Immunodominance is recognized as a key factor in the antigenic drift of seasonal influenza viruses. In the immunodominance model, each individual in a population predominantly responds to a single epitope among the five antigenic epitopes of the viral hemagglutinin (HA), driving escape mutations one at a time, and sequential mutations in multiple individuals who respond to different epitopes eventually generate a drifted strain with mutations in epitopes that are targeted by a majority of the population. A focused antibody response to the Sa epitope in people born between 1965 and 1979 was believed to contribute to a mutation at HA residue 163 and the first antigenic drift of the 2009 pandemic influenza A H1N1 virus. A serine-to-threonine mutation at HA residue 185 in the Sb epitope emerged in 2010 even before the 163 mutation. We show here that a large fraction of the population in 2010-2011 had responses to the Sb epitope, as shown by 47% of tested sera having altered titers to the S185T mutant. Responses to the Sb epitope showed an age-specific trend similar to that found for the response to Sa epitope in these subjects. Together, the focused responses to Sa and Sb epitopes may have driven the first antigenic drift of the 2009 pandemic H1N1 virus.

Influence of cellular lipid content on influenza A virus replication.

Influenza A virus (IAV) depends on the metabolism of its cellular host to provide energy and essential factors, including lipids, for viral replication. Previous studies have shown that fatty acids (FAs) play an important role in IAV replication and that inhibition of FA biosynthesis can diminish viral replication. However, cellular lipids can either be synthesized intracellularly or be imported from the extracellular environment. Interfering with FA import mechanisms may reduce the cellular lipid content and inhibit IAV replication. To test this hypothesis, MDCK and Detroit 562 cells were infected with IAV followed by exposure to palmitic acid and inhibitors of FA import. Replication of IAV significantly increased when infected cells were supplied with palmitic acid. This enhancement could be reduced by adding an FA import inhibitor. The addition of palmitic acid significantly increased the cellular lipid content, and this increased level was reduced by treatment with an FA import inhibitor. These results show that reducing the cellular lipid level might be an approach for IAV therapy.

The relationship of codon usage to the replication strategy of parvoviruses.

Codon usage is biased in most species, and the pattern of codon usage bias is specific to each species or group of closely related species. Although viruses use the host translational machinery for synthesis of their proteins, their codon usage patterns do not match those of their host. Viral codon usage is determined by a complex interplay of mutational bias, genome composition constraints, translational adaptation to the host, and host cellular innate defense. The codon usage of parvoviruses was previously shown not to be strongly biased and selective pressure was found to be a dominating factor driving codon usage. The family Parvoviridae includes the genus Dependoparvovirus, some of the members of which require a helper virus to complete their replication cycle, whereas the rest of the family can replicate without the need for helper viruses. Here, we show that difference in the replication strategy of these viruses may be an important factor determining viral codon usage. Hierarchical clustering and principal component analysis revealed that the codon usage pattern of adeno-associated viruses (AAVs) of the genus Dependoparvovirus is distinct from that of members of the other genera of vertebrate parvoviruses, and even from that of independent viruses of the genus Dependoparvovirus. Furthermore, the codon usage of human AAVs was found to be similar to that of some human adenoviruses in hierarchical clustering and principal component analysis. This suggests that the codon usage of AAVs is different from that of other parvoviruses because of their distinctive replication strategy and that their codon usage is probably driven by forces similar to those that shaped the codon usage pattern of their helper viruses.

Genome polarity of RNA viruses reflects the different evolutionary pressures shaping codon usage.

RNA viruses are classified by their genome polarity and replication strategies. Nucleotide composition and codon usage differ among virus groups, for instance positive-sense RNA (+ssRNA) viruses have higher GC-content than the other RNA virus groups. Codon usage of +ssRNA viruses is closer to humans showing significantly higher codon adaptation index (CAI) than those of negative-sense RNA (-ssRNA), double stranded RNA (dsRNA) and retroviruses. Ambisense viruses have high CAI comparable to that of +ssRNA virus despite their lower GC content, whereas dsRNA viruses have the lowest CAI. This may provide a benefit for +ssRNA viruses as their genomes are used as mRNA. However, analyses for influence of nucleotide composition on codon usage did not show a difference between +ssRNA and -ssRNA viruses. This suggests that genome composition and hence mutational pressure remain the major pressure causing the differences in codon usage among RNA viruses with different genome types.

Codon usage of HIV regulatory genes is not determined by nucleotide composition.

Codon usage bias can be a result of either mutational bias or selection for translational efficiency and/or accuracy. Previous data has suggested that nucleotide composition constraint was the main determinant of HIV codon usage, and that nucleotide composition and codon usage were different between the regulatory genes, tat and rev, and other viral genes. It is not clear whether translational selection contributed to the codon usage difference and how nucleotide composition and translational selection interact to determine HIV codon usage. In this study, a model of codon bias due to GC composition with modification for the A-rich third codon position was used to calculate predicted HIV codon frequencies based on its nucleotide composition. The predicted codon usage of each gene was compared with the actual codon frequency. The predicted codon usage based on GC composition matched well with the actual codon frequencies for the structural genes (gag, pol and env). However, the codon usage of the regulatory genes (tat and rev) could not be predicted. Codon usage of the regulatory genes was also relatively unbiased showing the highest effective number of codons (ENC). Moreover, the codon adaptation index (CAI) of the regulatory genes showed better adaptation to human codons when compared to other HIV genes. Therefore, the early expressed genes responsible for regulation of the replication cycle, tat and rev, were more similar to humans in terms of codon usage and GC content than other HIV genes. This may help these genes to be expressed efficiently during the early stages of infection.

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid.

UNLABELLED: Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE: Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.

Exposure to cold impairs interferon-induced antiviral defense.

It is commonly believed that exposure to low temperature increases susceptibility to viral infection in the human respiratory tract, but a molecular mechanism supporting this belief has yet to be discovered. In this study, we investigated the effect of low temperature on viral infection and innate defense in cell lines from the human respiratory tract and found that interferon-induced antiviral responses were impaired at low temperatures. Cells maintained at 25°C and 33°C expressed lower levels of myxovirus resistance protein 1 (MxA) and 2'5'-oligoadenylate synthetase 1 (OAS1) mRNAs when compared to cells maintained at 37°C after infection by seasonal influenza viruses. Exogenous ß-interferon treatment reduced the viral replication at 37°C, but not at 25°C. Our results suggest that the impairment of interferon-induced antiviral responses by low temperature is one of several mechanisms that could explain an increase in host susceptibility to respiratory viruses after exposure to cold temperature.

Expression of importin-α isoforms in human nasal mucosa: implication for adaptation of avian influenza A viruses to human host.

BACKGROUND: Transportation into the host cell nucleus is crucial for replication and transcription of influenza virus. The classical nuclear import is regulated by specific cellular factor, importin-α. Seven isoforms of importin-α have been identified in human. The preference of importin-α3 of avian influenza virus and -α7 isoform of human strains during replication in human cells was previously identified. In addition, both avian and human influenza viruses were shown to use importin-α1 isoform for their replication. FINDING: The mRNA levels of importin-α1, -α3, and -α7 isoforms in human respiratory tract was determined by real-time RT-PCR. The results indicate that mRNA level of importin-α7 was significantly higher than that of importin-α1 (p-value < 0.0001) and importin-α3 (p-value < 0.0001) isoforms in human nasal mucosa while importin-α1 was detected as the highest expression importin-α isoform in lung tissues. CONCLUSIONS: These results may explain the preference of importin-α7 isoforms in seasonal influenza viruses in human upper respiratory tract and may suggest a selective pressure toward importin-α7 in human respiratory tract infection of an avian virus.

Sialic acid content in human saliva and anti-influenza activity against human and avian influenza viruses.

It was shown previously that human saliva has higher antiviral activity against human influenza viruses than against H5N1 highly pathogenic avian influenza viruses, and that the major anti-influenza activity was associated with sialic-acid-containing molecules. To further characterize the differential susceptibility to saliva among influenza viruses, seasonal influenza A and B virus, pandemic H1N1 virus, and 15 subtypes of avian influenza virus were tested for their susceptibility to human and chicken saliva. Human saliva showed higher hemagglutination inhibition (HI) and neutralization (NT) titers against seasonal influenza A virus and the pandemic H1N1 viruses than against influenza B virus and most avian influenza viruses, except for H9N2 and H12N9 avian influenza viruses, which showed high HI and NT titers. To understand the nature of sialic-acid-containing anti-influenza factors in human saliva, α2,3- and α2,6-linked sialic acid was measured in human saliva samples using a lectin binding and dot blot assay. α2,6-linked sialic acid was found to be more abundant than α2,3-linked sialic acid, and a seasonal H1N1 influenza virus bound more efficiently to human saliva than an H5N1 virus in a dot blot analysis. These data indicated that human saliva contains the sialic acid type corresponding to the binding preference of seasonal influenza viruses.

The N-linked glycosylation site at position 158 on the head of hemagglutinin and the virulence of H5N1 avian influenza virus in mice.

N-linked glycosylation of the influenza virus hemagglutinin (HA) protein plays crucial roles in HA structure and function, evasion of neutralizing antibodies, and susceptibility to innate soluble antiviral factors. The N-linked glycosylation site at position 158 of highly pathogenic H5N1 virus was previously shown to affect viral receptor-binding preference. H5N1 viruses show heterogeneity with respect to the presence of this glycosylation site. Clade 1 viruses that caused outbreaks in Southeast Asia in 2004 contained this glycosylation site, while the site is absent in the more recent clade 2 viruses. Here, we show that elimination of this glycosylation site increases viral virulence in mice. The mutant lacking the glycosylation site at position 158 showed unaltered growth kinetics in vitro and a comparable level of sensitivity to a major antiviral protein found in respiratory secretions, surfactant protein D (SP-D).

Susceptibility of human and avian influenza viruses to human and chicken saliva.

Oral cavity can be an entry site of influenza virus and saliva is known to contain innate soluble anti-influenza factors. Influenza strains were shown to vary in their susceptibility to those antiviral factors. Whether the susceptibility to the saliva antiviral factors plays any role in the host species specificity of influenza viruses is not known. In this study, the antiviral activity of human and chicken saliva against human and the H5N1 avian influenza viruses were investigated by hemagglutination inhibition (HI) and neutralization (NT) assays. In comparison to human influenza viruses, H5N1 isolates showed reduced susceptibility to human saliva as measured by HI and NT assays. Interestingly, an H5N1 isolate that bind to both α2,3- and α2,6-linked sialic acid showed much higher HI titers with human saliva, suggesting that the susceptibility profile was linked to the receptor-binding preference and the presence of α2,6-linked sialic in human saliva. On the other hand, the H5N1 isolates showed increased HI titers but reduced NT titers to chicken saliva as compared to human influenza isolates. The human salivary antiviral components were characterized by testing the sensitivity to heat, receptor destroying enzyme (RDE), CaCl2/EDTA dependence, and inhibition by mannan, and shown to be α- and γ-inhibitors. These data suggest that the H5N1 HPAI influenza virus had distinctive susceptibility patterns to human and chicken saliva, which may play some roles in its infectivity and transmissibility in these hosts.

Antibody Response to Influenza Hemagglutinin Conserved Stalk Domain after Sequential Immunization with Old Vaccine Strains.

Hemagglutinin (HA) is the major envelope glycoprotein and antigen on the surface of influenza virions. The glycoprotein comprises a globular head and a stalk region. While immunodominant epitopes on influenza HA head are highly variable, the stalk domain is conserved. The variability of the HA head causes the antigenic drift that made the requirement of annual update of vaccine strains. Induction of antibody against the stalk domain has been proposed as an approach for a broadly protective influenza vaccine strategy. Sequential exposure to influenza strains with highly diverse HA heads but conserved stalks have been shown to induce antibody to the low immunogenic stalk domain. Here, we tested this approach by using old influenza vaccine strains that are decades apart in evolution. Inactivated whole virion vaccine of influenza A/Puerto Rico/8/1934, A/USSR/92/1977, and A/Thailand/102/2009 (H1N1) was sequentially immunized into BALB/c mice in comparison to immunization using single strain (A/Thailand/102/2009 (H1N1)). The sequentially immunized mice developed higher levels of binding antibody to the stalk domain. These suggested that using old vaccine strains in sequential vaccination may be a possible approach to induce antibody to the conserved stalk domain.

A serine-to-asparagine mutation at position 314 of H5N1 avian influenza virus NP is a temperature-sensitive mutation that interferes with nuclear localization of NP.

We have generated a temperature-sensitive (ts) mutant from a human isolate of the H5N1 avian influenza virus by classical adaptation in cell culture. After 20 passages at low temperature, the virus showed a ts phenotype. The ts mutant also showed an attenuated phenotype after nasal inoculation in mice. Using reverse genetics, we generated reassortants carrying individual genomic segments of the wild-type and mutant viruses in an A/Puerto Rico/8/34 background, and found that the nucleoprotein (NP) gene could confer the ts phenotype. This mutant NP contains a serine-to-asparagine mutation at position 314 (S314N). The mutant NP protein showed a defect in nuclear localization at high temperature in mammalian cells.

Human Schlafen 11 inhibits influenza A virus production.

Schlafen (SLFN) proteins are a subset of interferon-stimulated early response genes with antiviral properties. An antiviral mechanism of SLFN11 was previously demonstrated in human immunodeficiency virus type 1 (HIV-1)-infected cells, and it was shown that SLFN11 inhibited HIV-1 virus production in a codon usage-specific manner. The codon usage patterns of many viruses are vastly different from those of their hosts. The codon usage-specific inhibition of HIV-1 expression by SLFN11 suggests that SLFN11 may be able to inhibit other viruses with a suboptimal codon usage pattern. However, the effect of SLFN11 on the replication of influenza A virus (IAV) has never been reported. The induction of SLFN11 expression was observed upon IAV infection. The reduction of SLFN11 expression also promotes influenza virus replication. Moreover, we found that overexpression of SLFN11 could reduce the expression of a reporter gene with a viral codon usage pattern, and the inhibition of viral hemagglutinin (HA) gene was codon-specific as the expression of codon optimized HA was not affected. These results indicate that SLFN11 inhibits the influenza A virus in a codon-specific manner and that SLFN11 may contribute to innate defense against influenza A viruses.

Efficacy of combined COVID-19 convalescent plasma with oral RNA-dependent RNA polymerase inhibitor treatment versus neutralizing monoclonal antibody therapy in COVID-19 outpatients: a multi-center, non-inferiority, open-label randomized controlled trial (PlasMab).

IMPORTANCE: This pivotal study reveals that high neutralizing titer COVID-19 convalescent plasma therapy (CPT) combined with favipiravir (FPV) is non-inferior to sotrovimab in preventing hospitalization and severe outcomes in outpatients with mild-to-moderate COVID-19 and high-risk comorbidities. It underscores the potential of CPT-FPV as a viable alternative to neutralizing monoclonal antibodies like sotrovimab, especially amid emerging variants with spike protein mutations. The study's unique approach, comparing a monoclonal antibody with CPT, demonstrates the efficacy of early intervention using high neutralizing antibody titer CPT, even in populations with a significant proportion of elderly patients. These findings are crucial, considering the alternative treatment challenges, especially in resource-limited countries, posed by the rapidly mutating SARS-CoV-2 virus and the need for adaptable therapeutic strategies.

Analysis of Influenza A virus infection in human induced pluripotent stem cells (hiPSCs) and their derivatives.

Influenza A virus (IAV) infection in pregnant women is a major public health concern. However, the effect of IAV infection on human embryogenesis is still unclear. Here we show that human induced pluripotent stem cells (hiPSCs) and hiPSC-derived ectodermal, mesodermal and endodermal cells are susceptible to IAV infection. These cell types stained positive for α2,6-linked sialic acid, the receptor for IAV infection expressed on the cell surface. While hiPSCs produced high viral titers for up to 7 days with increasing infected cell number suggesting that the viral progenies produced from hiPSCs without exogenous protease were infectious and could spread to other cells, the three germ-layer cells showed a decline in viral titers suggesting the lack of viral spreading. Amongst the three germ layers, endodermal cells were less susceptible than ectodermal and mesodermal cells. These results indicate the permissiveness of cells of early embryogenesis, and suggest a risk of detrimental effects of IAV infection in early human embryonic development.

Case report: Increased viral receptor expression associated with high viral load and severe pneumonia in a young patient infected with 2009 H1N1 influenza a with no pre-existing conditions.

A case of unusually high severity of influenza pneumonia leading to acute respiratory distress syndrome and death was investigated. This was a previously a healthy 28-year-old man with no underlying conditions, admitted to a hospital during the first wave of influenza pandemic in Thailand in July 2009. He had experienced high fever and influenza-like illness for 5 days before coming to the hospital. He developed acute respiratory distress syndrome and expired on day 7 after admission. In comparison to three other cases of influenza pneumonia in the same outbreak with known risk factors for severe influenza, such as pregnancy and diabetes mellitus, a much higher viral load was detected in the lungs of this patient despite antiviral treatment. In agreement with the high viral load, the lung specimens from this patient, but not the other three patients, showed a high expression of α-2,6-linked sialic acid by lectin staining. The gene responsible for the synthesis of this sialic acid was also found to be upregulated. The data indicated overexpression of the viral receptor as a potential mechanism for severe disease in some patients.

SARS-CoV-2 seroprevalence among cancer patients and household caregivers in a cancer hospital: Cross-sectional survey after the second COVID-19 wave in Thailand.

Objectives: Patients with cancer may be at an increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and experience more severe outcomes. Low vaccine coverage in the early phase of the coronavirus disease 2019 (COVID-19) pandemic meant that personal and social measures to reduce viral spread were the only methods of lowering the risk of infection among cancer patients. This study explored the prevalence of SARS-CoV-2 antibodies in cancer patients and caregivers in a cancer hospital after the second COVID-19 outbreak in Thailand. Study design: Cross-sectional study. Methods: A SARS-CoV-2 seroprevalence cross-sectional survey was conducted among 200 cancer patients and 200 household caregivers in a tertiary cancer care hospital in Bangkok, Thailand. The survey took place between 4 March and May 31, 2021 - a time period covering the end of the second COVID-19 wave and the early phase of the third wave in Thailand. Results: Rigorous personal and social measures to reduce viral spread among cancer patients and caregivers lead to an extremely low prevalence of SARS-CoV2 infection (0% among cancer patients and 1% among household caregivers). Conclusion: This study demonstrates the importance of social distancing and personal hygiene measures for the prevention of SARS-CoV-2 infection, even when vaccine coverage is low.

Viral load of the highly pathogenic avian influenza H5N1 virus in infected human tissues.

The highly pathogenic avian influenza A (H5N1) virus is a virulent virus that causes an acute febrile respiratory disease with high mortality in humans. To gain a better insight of H5N1 viral distributions in infected human tissues, the levels of viral RNA were determined in the autopsy tissues from two patients who were infected with H5N1 virus by using real-time reverse transcription-polymerase chain reaction. In one patient who died on day 6 of the illness, the viral load in the lung was extremely high, whereas the levels of viral RNA in the other organs were more than 6 log lower. In the other patient who died on day 17 of the illness, the viral load was similar in the lung and other organs, and was comparable to the viral load in the extra-pulmonary tissues of the first patient. These results suggested that while the H5N1 virus can cause disseminated infection in humans, the lung is still the major site of viral replication, and viral replication in the lung in the later stages may decrease as a result of the depletion of the available target cells. In addition, the mRNA levels of the tumor necrosis factor-α (TNF-α) were found to be associated with the viral titers.