Towards broad-spectrum protection: the development and challenges of combined respiratory virus vaccines.

In the post-COVID-19 era, the co-circulation of respiratory viruses, including influenza, SARS-CoV-2, and respiratory syncytial virus (RSV), continues to have significant health impacts and presents ongoing public health challenges. Vaccination remains the most effective measure for preventing viral infections. To address the concurrent circulation of these respiratory viruses, extensive efforts have been dedicated to the development of combined vaccines. These vaccines utilize a range of platforms, including mRNA-based vaccines, viral vector vaccines, and subunit vaccines, providing opportunities in addressing multiple pathogens at once. This review delves into the major advancements in the field of combined vaccine research, underscoring the strategic use of various platforms to tackle the simultaneous circulation of respiratory viruses effectively.

The Impact of Medical Resources and Oral Antiviral Drugs on SARS-CoV-2 Mortality - Hong Kong SAR, China, 2022.

Introduction: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates increased transmissibility compared to earlier strains, contributing to a significant number of fatalities in Hong Kong Special Administrative Region (HKSAR), China. Adequate medical resources and medications are essential in mitigating these deaths. This study evaluates the effects of supplementary resources from the Chinese mainland during the fifth wave of the pandemic in HKSAR. Methods: Vector autoregression (VAR) was employed to analyze data from the Oxford coronavirus disease 2019 (COVID-19) Government Response Tracker to assess the effectiveness of control measures during five waves of the pandemic in HKSAR. Additionally, a transmission dynamics model was created to investigate the influence of supplementary medical resources from the Chinese mainland and oral medications on mortality. Results: In the initial four waves, workplace closures, restrictions on public events, international travel bans, and shielding the elderly significantly influenced pandemic management. Contrarily, during the fifth wave, these measures showed no notable effects. When comparing a situation without extra medical resources or COVID-19 oral medication, there was a 17.7% decrease in COVID-19 fatalities with mainland medical resources and an additional 10.2% reduction with oral medications. Together, they contributed to a 26.6% decline in fatalities. Discussion: With the rapid spread of the virus, regional reallocation of medical resources may reduce mortality even when the local healthcare system is overstretched.

Seroprevalence of Avian Influenza A(H5N6) Virus Infection, Guangdong Province, China, 2022.

In 2022, we assessed avian influenza A virus subtype H5N6 seroprevalence among the general population in Guangdong Province, China, amid rising numbers of human infections. Among the tested samples, we found 1 to be seropositive, suggesting that the virus poses a low but present risk to the general population.

Engineering of novel hemagglutinin biosensors for rapid detection and drug screening of Influenza A H7N9 virus.

New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.

Baloxavir marboxil use for critical human infection of avian influenza A H5N6 virus.

BACKGROUND: Recent outbreaks of avian influenza and ongoing virus reassortment have drawn focus on spill-over infections. The increase in human infections with highly pathogenic avian influenza H5N6 virus and its high fatality rate posed a potential threat, necessitating the search for a more effective treatment. METHODS: Longitudinal clinical data and specimens were collected from five H5N6 patients after admission. All patients received antiviral treatment of either sequential monotherapy of oseltamivir and baloxavir or the two drugs in combination. Severity of illness; viral load in sputum, urine, and blood; and cytokine levels in serum and sputum were serially analyzed. FINDINGS: All patients developed acute respiratory distress syndrome (ARDS) and viral sepsis within 1 week after disease onset. When delayed oseltamivir showed poor effects, baloxavir was administered and rapidly decreased viral load. In addition, levels of IL-18, M-CSF, IL-6, and HGF in sputum and Mig and IL-18 in serum that reflected ARDS and sepsis deterioration, respectively, were also reduced with baloxavir usage. However, three patients eventually died from exacerbation of underlying disease and secondary bacterial infection. Nonsurvivors had more severe extrapulmonary organ dysfunction and insufficient H5N6 virus-specific antibody response. CONCLUSIONS: For critical human cases of H5N6 infection, baloxavir demonstrated effects on viral load and pulmonary/extrapulmonary cytokines, even though treatment was delayed. Baloxavir could be regarded as a first-line treatment to limit continued viral propagation, with potential future application in avian influenza human infections and poultry workers exhibiting influenza-like illness. FUNDING: This work was funded by the National Natural Science Foundation of China (81761128014).

Influenza Virus Carrying a Codon-Reprogrammed Neuraminidase Gene as a Strategy for Live Attenuated Vaccine.

Live attenuated influenza vaccines offer broader and longer-lasting protection in comparison to inactivated influenza vaccines. The neuraminidase (NA) surface glycoprotein of influenza A virus is essential for the release and spread of progeny viral particles from infected cells. In this study, we de novo synthesized the NA gene, in which 62% of codons were synonymously changed based on mammalian codon bias usage. The codon-reprogrammed NA (repNA) gene failed to be packaged into the viral genome, which was achievable with partial restoration of wild-type NA sequence nucleotides at the 3' and 5' termini. Among a series of rescued recombinant viruses, we selected 20/13repNA, which contained 20 and 13 nucleotides of wild-type NA at the 3' and 5' termini of repNA, respectively, and evaluated its potential as a live attenuated influenza vaccine. The 20/13repNA is highly attenuated in mice, and the calculated LD50 was about 10,000-fold higher than that of the wild-type (WT) virus. Intranasal inoculation of the 20/13repNA virus in mice induced viral-specific humoral, cell-mediated, and mucosal immune responses. Mice vaccinated with the 20/13repNA virus were protected from the lethal challenge of both homologous and heterologous viruses. This strategy may provide a new method for the development of live, attenuated influenza vaccines for a better and more rapid response to influenza threats.

Active immunization with Pseudomonas aeruginosa vaccine protects mice from secondary Pseudomonas aeruginosa challenge post-influenza virus infection.

Background: Influenza virus infection complicated by secondary bacterial pneumonia contributes significantly to death during seasonal or pandemic influenza. Secondary infection of Pseudomonas aeruginosa (P. aeruginosa) in influenza virus-infected patients contributes to morbidity and mortality. Methods: Mice were first infected with PR8 influenza virus, followed by a secondary infection of P. aeruginosa. Body weights and survival rate of mice was monitored daily over 20 days. Bronchoalveolar lavage fluids (BALFs) and lung homogenates were harvested for measuring bacterial titers. Lung tissue section slides were stained with hematoxylin and eosin for microscopic observation. After vaccination with inactivated P. aeruginosa cells or recombinant PcrV protein, the mice were subjected to PR8 influenza virus infection followed by a secondary infection of a P. aeruginosa. The inhibition against P. aeruginosa of serum was evaluated by detecting the growth of P. aeruginosa in broth containing diluted sera. Results: The prior influenza infection greatly enhanced the susceptibility to secondary infection of P. aeruginosa and increased morbidity and mortality in mice. Active immunization with inactivated P. aeruginosa cells could protect mice from secondary P. aeruginosa challenge in influenza virus infected mice. Conclusions: To develop an effective P. aeruginosa vaccine might be a promising strategy to decrease the threat of secondary P. aeruginosa infection in influenza patients.

Immunization with a Prefusion SARS-CoV-2 Spike Protein Vaccine (RBMRNA-176) Protects against Viral Challenge in Mice and Nonhuman Primates.

There is an urgent need for a broad-spectrum and protective vaccine due to the emergence and rapid spreading of more contagious SARS-CoV-2 strains. We report the development of RBMRNA-176, a pseudouridine (Ψ) nucleoside-modified mRNA-LNP vaccine encoding pre-fusion stabilized trimeric SARS-CoV-2 spike protein ectodomain, and evaluate its immunogenicity and protection against virus challenge in mice and nonhuman primates. A prime-boost immunization with RBMRNA-176 at intervals of 21 days resulted in high IgG titers (over 1:819,000 endpoint dilution) and a CD4+ Th1-biased immune response in mice. RBMRNA-176 vaccination induced pseudovirus-neutralizing antibodies with IC50 ranging from 1:1020 to 1:2894 against SARS-CoV-2 spike pseudotyped wild-type and variant viruses, including Alpha, Beta, Gamma, and Kappa. Moreover, significant control of viral replication and histopathology in lungs was observed in vaccinated mice. In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the prime induced a persistent and sustained IgG response. RBMRNA-176 vaccination also protected macaques against upper and lower respiratory tract infection, as well as lung injury. Altogether, these findings support RBMRNA-176 as a vaccine candidate for prevention of COVID-19.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.

L226Q Mutation on Influenza H7N9 Virus Hemagglutinin Increases Receptor-Binding Avidity and Leads to Biased Antigenicity Evaluation.

Since the first outbreak in 2013, the influenza A (H7N9) virus has continued emerging and has caused over five epidemic waves. Suspected antigenic changes of the H7N9 virus based on hemagglutination inhibition (HI) assay during the fifth outbreak have prompted the update of H7N9 candidate vaccine viruses (CVVs). In this study, we comprehensively compared the serological cross-reactivities induced by the hemagglutinins (HAs) of the earlier CVV A/Anhui/1/2013 (H7/AH13) and the updated A/Guangdong/17SF003/2016 (H7/GD16). We found that although H7/GD16 showed poor HI cross-reactivity to immune sera from mice and rhesus macaques vaccinated with either H7/AH13 or H7/GD16, the cross-reactive neutralizing antibodies between H7/AH13 and H7/GD16 were comparably high. Passive transfer of H7/AH13 immune sera also provided complete protection against the lethal challenge of H7N9/GD16 virus in mice. Analysis of amino acid mutations in the HAs between H7/AH13 and H7/GD16 revealed that L226Q substitution increases the HA binding avidity to sialic acid receptors on red blood cells, leading to decreased HI titers against viruses containing HA Q226 and thus resulting in a biased antigenic evaluation based on HI assay. These results suggest that amino acids located in the receptor-binding site could mislead the evaluation of antigenic variation by solely impacting the receptor-binding avidity to red blood cells without genuine contribution to antigenic drift. Our study highlighted that viral receptor-binding avidity and combination of multiple serological assays should be taken into consideration in evaluating and selecting a candidate vaccine virus of H7N9 and other subtypes of influenza viruses.IMPORTANCE The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using the HI assay. Other serological assays, such as the microneutralization (MN) assay, should be considered a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.

An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques.

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.

Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

Evaluation of the immune response of a H7N9 candidate vaccine virus derived from the fifth wave A/Guangdong/17SF003/2016.

Highly pathogenic influenza H7N9 viruses that emerged in the fifth wave of H7N9 outbreak pose a risk to human health. The World Health Organization has updated the candidate vaccine viruses for H7N9 viruses recently. In this study, we evaluated the immune response to an updated H7N9 candidate vaccine virus, which derived from the highly pathogenic A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. GD/16 vaccination elicited robust neutralizing, virus-specific immunoglobulin G antibodies and effective protection, but poor hemagglutination inhibition antibody titers. Furthermore, mouse and rhesus macaque serum raised against the previous H7N9 CVV A/Anhui/1/2013 (AH/13) were tested for its cross-reactivity to GD/16 virus. We found that although AH/13-immune serum has poor hemagglutination inhibition reactivity against GD/16 virus, AH/13 elicit efficient cross-neutralizing antibodies and in vivo protection against GD/16. Further studies showed that the hemagglutinin of GD/16 has strong receptor binding avidity, which might be associated with the decreased hemagglutination inhibition assay sensitivity. This study underscores the point that receptor binding avidity should be taken into account when performing quantitative interpretation of hemagglutination inhibition data. A combination of multiple serological assays is required for accurate vaccine evaluation and antigenic analysis of influenza viruses.

Phenotypic Characterization of Chinese Rhesus Macaque Plasmablasts for Cloning Antigen-Specific Monoclonal Antibodies.

Rhesus macaques (Macaca mulatta) are used as a human-relevant animal species for the evaluation of vaccines and as a source for cloning monoclonal antibodies (mAbs) that are highly similar to human-derived antibodies. Although antibody-secreting plasmablasts in humans are well-defined and can be easily isolated for mAb cloning, it remains unclear whether the same phenotypic markers could be applied for isolating antibody-secreting plasmablasts from Chinese rhesus macaques. In this study, we evaluated a series of cell surface and intracellular markers and identified the phenotypic markers of plasmablasts in Chinese rhesus macaques as CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+. After influenza virus vaccination, the plasmablasts in peripheral blood mononuclear cells (PBMCs) increased transiently, peaked at day 4-7 after booster vaccination and returned to nearly undetectable levels by day 14. Antigen-specific enzyme-linked immunosorbent spot (ELISPOT) assays confirmed that the majority of the plasmablasts could produce influenza virus-specific antibodies. These plasmablasts showed transcriptional characteristics similar to those of human plasmablasts. Using single-cell PCR for immunoglobulin heavy and light chains, most mAbs cloned from the CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+ plasmablasts after vaccination exhibited specific binding to influenza virus. This study defined the phenotypic markers for isolating antibody-secreting plasmablasts from Chinese rhesus macaques, which has implications for efficient cloning of mAbs and for the evaluation of plasmablast response after vaccination or infection in Chinese rhesus macaques.

Chloroquine inhibits endosomal viral RNA release and autophagy-dependent viral replication and effectively prevents maternal to fetal transmission of Zika virus.

Zika virus (ZIKV) infection can cause neonatal microcephaly and neurological disorders. Currently, there is no designated drug for treating ZIKV infection and preventing neonatal microcephaly. In this study, we evaluated the effect of chloroquine, an anti-malaria drug, in ZIKV infected cells and mouse models. Chloroquine significantly inhibited ZIKV infection in multiple mammalian cell lines. Chloroquine treatment significantly improved the survival of ZIKV-infected 1-day old suckling SCID Beige mice and reduced viremia in adult SCID Beige mice. Importantly, chloroquine protected the fetus from maternal infection by reducing placenta to fetus viral transmission. We found that chloroquine exerts at least two mechanisms in protecting against ZIKV infection: 1) inhibiting endosomal disassembly of the internalized virus and thus reducing the release of viral RNA to the cytoplasm for replication; 2) inhibiting ZIKV RNA replication through blocking ZIKV induced autophagy. Our study suggests that chloroquine treatment warrants to be considered as a mitigation strategy for treating ZIKV infection and preventing ZIKV-associated microcephaly in pregnant women.

Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model.

The Zika virus (ZIKV) outbreak and its link to microcephaly triggered a public health concern. To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 heavy and light chain-paired monoclonal antibodies (mAbs) that bind to ZIKV envelope (E) proteins isolated from memory B cells of a ZIKV-infected patient. Three mAbs (7B3, 1C11, and 6A6) that showed the most potent and broad neutralization activities against the African, Asian, and American strains were selected for further analysis. mAb 7B3 showed an IC50 value of 11.6 ng/mL against the circulating American strain GZ02. Epitope mapping revealed that mAbs 7B3 and 1C11 targeted residue K394 of the lateral ridge (LR) epitope of the EDIII domain, but 7B3 has a broader LR epitope footprint and recognizes residues T335, G337, E370, and N371 as well. mAb 6A6 recognized residues D67, K118, and K251 of the EDII domain. Interestingly, although the patient was seronegative for DENV infection, mAb 1C11, originating from the VH3-23 and VK1-5 germline pair, neutralized both ZIKV and DENV1. Administration of the mAbs 7B3, 1C11, and 6A6 protected neonatal SCID mice infected with a lethal dose of ZIKV. This study provides potential therapeutic antibody candidates and insights into the antibody response after ZIKV infection.

Development and application of bioluminescence imaging for the influenza A virus.

Influenza A viruses (IAVs) cause seasonal epidemics and intermittent pandemics which threaten human health. Conventional assays cannot meet the demands for rapid and sensitive detection of viral spread and pathogenesis in real time cannot be used for high-throughput screens of novel antivirals. Bioluminescence imaging (BLI) has emerged as a powerful tool in the study of infectious diseases in animal models. The advent of influenza reverse genetics has enabled the incorporation of bioluminescent reporter proteins into replication-competent IAVs. This review briefly describes the current development and applications of bioluminescence in the study of viral infections and antiviral therapeutics for IAVs. BLI is expected to substantially accelerate the basic and applied research of IAV both in vitro and in vivo.

Incorporation of NS1 and prM/M are important to confer effective protection of adenovirus-vectored Zika virus vaccine carrying E protein.

Current design of Zika virus (ZIKV) vaccine mainly considered envelope (E) as the major target antigen. Non-structural protein NS1 was seldom considered. Herein, we generated three adenovirus-vectored vaccines carrying E (Ad2-E), or premembrane/membrane (prM/M) with E (Ad2-prME), or NS1 in addition to prM/M with E (Ad2-prME-NS1). Ad2-prME induced higher neutralizing antibody response to ZIKV than Ad2-E, suggesting prM/M is important for the folding of immunogenic E. Most intriguingly, Ad2-prME-NS1 elicited the best viral inhibition when the immune sera were added to ZIKV-infected cells. In ZIKV-challenged neonatal mice born to maternally immunized dams, Ad2-prME-NS1 conferred the best protection in preventing weight loss, neurological disorders, and viral replication. Ad2-prME also conferred significant protection but was less effective than Ad2-prME-NS1, whereas Ad2-E only alleviated neurological symptoms but did not inhibit viral replication. Our study suggested that NS1 should be considered in the design of ZIKV vaccine in addition to prM/M and E.

Patient-derived avian influenza A (H5N6) virus is highly pathogenic in mice but can be effectively treated by anti-influenza polyclonal antibodies.

Highly pathogenic avian influenza A (H5N6) virus has been circulating in poultry since 2013 and causes sporadic infections and fatalities in humans. Due to the re-occurrence and continuous evolution of this virus subtype, there is an urgent need to better understand the pathogenicity of the H5N6 virus and to identify effective preventative and therapeutic strategies. We established a mouse model to evaluate the virulence of H5N6 A/Guangzhou/39715/2014 (H5N6/GZ14), which was isolated from an infected patient. BALB/c mice were inoculated intranasally with H5N6/GZ14 and monitored for morbidity, mortality, cytokine production, lung injury, viral replication, and viral dissemination to other organs. H5N6/GZ14 is highly pathogenic and can kill 50% of mice at a very low infectious dose of 5 plaque-forming units (pfu). Infection with H5N6/GZ14 showed rapid disease progression, viral replication to high titers in the lung, a strongly induced pro-inflammatory cytokine response, and severe lung injury. Moreover, infectious H5N6/GZ14 could be detected in the heart and brain of the infected mice. We also demonstrated that anti-influenza polyclonal antibodies generated by immunizing rhesus macaques could protect mice from lethal infection. Our results provide insights into the pathogenicity of the H5N6 human isolate.