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Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology., (© 2024. Battelle Memorial Institute.)

Besides the common receptor/coreceptor-dependent mechanism of cellular attachment, some viruses rely on antiviral antibodies for their efficient entry into target cells. This mechanism, known as antibody-dependent enhancement (ADE) of viral infection, depends on the cross-linking of complexes of virus-antibody or virus-activated complement components through interaction with cellular molecules such as Fc receptors or complement receptors, leading to enhanced infection of susceptible cells. Recent studies have suggested that additional mechanisms underlie ADE: involvement of complement component C1q and its receptor (Ebola virus), antibody-mediated modulation of the interaction between viral protein and its coreceptor (human immunodeficiency virus) and suppression of cellular antiviral genes by the replication of viruses entering cells via ADE (Ross River virus). Since ADE is exploited by a variety of viruses and has been associated with disease exacerbation, it may have broad relevance to the pathogenesis of viral infection and antiviral strategies., (Copyright 2003 John Wiley & Sons, Ltd.)

Background: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long‐term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown. Methods: We established a duplex real‐time RT‐PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap‐dependent endonuclease inhibitor baloxavir marboxil. Results: We detected three influenza C viruses belonging to the C/Kanagawa‐ or C/Sao Paulo‐lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa‐lineage, showed similar antigenicity to the reference C/Kanagawa‐lineage strain and was susceptible to baloxavir. Conclusions: Our duplex real‐time RT‐PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus. [ABSTRACT FROM AUTHOR]

Vaccination is an efficient approach to preventing influenza virus infections. Recently, we developed influenza A and B virus vaccine backbones that increased the yield of several vaccine viruses in Madin–Darby canine kidney (MDCK) and African green monkey kidney (Vero) cells. These vaccine backbones also increased viral replication in embryonated chicken eggs, which are the most frequently used platform for influenza vaccine manufacturing. In this study, to further increase the viral titers in embryonated chicken eggs, we introduced random mutations into the 'internal genes' (i.e., all influenza viral genes except those encoding the hemagglutinin and neuraminidase proteins) of the influenza A virus high-yield virus backbone we developed previously. The randomly mutated viruses were sequentially passaged in embryonated chicken eggs to select variants with increased replicative ability. We identified a candidate that conferred higher influenza virus growth than the high-yield parental virus backbone. Although the observed increases in virus growth may be considered small, they are highly relevant for vaccine manufacturers. [ABSTRACT FROM AUTHOR]

Objectives Widespread resistance of influenza viruses to neuraminidase (NA) inhibitor or polymerase inhibitor, baloxavir, is a major public health concern. The amino acid mutations R152K in NA and I38T in polymerase acidic (PA) are responsible for resistance to NA inhibitors and baloxavir, respectively. Methods We generated recombinant A(H1N1)pdm09 viruses possessing NA-R152K, PA-I38T or both mutations by using a plasmid-based reverse genetics system, characterized their virological properties in vitro and in vivo , and examined whether oseltamivir, baloxavir and favipiravir are effective against these mutant viruses. Results The three mutant viruses showed similar or superior growth kinetics and virulence to those of wild-type virus. Although oseltamivir and baloxavir blocked the replication of the wild-type virus in vitro , oseltamivir and baloxavir failed to suppress the replication of the NA-R152K and PA-I38T viruses in vitro , respectively. Mutant virus possessing both mutations grew in the presence of oseltamivir or baloxavir in vitro. Baloxavir treatment protected mice from lethal infection with wild-type or NA-R152K virus, but failed to protect mice from lethal infection with PA-I38T or PA-I38T/NA-R152K virus. Favipiravir treatment protected mice from lethal infection with all viruses tested, whereas oseltamivir treatment did not protect at all. Conclusions Our findings indicate that favipiravir should be used to treat patients with suspected baloxavir-resistant virus infection. [ABSTRACT FROM AUTHOR]

Routine surveillance in live poultry markets in the northern regions of Vietnam from 2016 to 2017 resulted in the isolation of 27 highly pathogenic avian H5N1 and H5N6 viruses of 3 different clades (2.3.2.1c, 2.3.4.4f, and 2.3.4.4g). Sequence and phylogenetic analysis of these viruses revealed reassortment with various subtypes of low pathogenic avian influenza viruses. Deep-sequencing identified minor viral subpopulations encoding variants that may affect pathogenicity and sensitivity to antiviral drugs. Interestingly, mice infected with two different clade 2.3.2.1c viruses lost body weight rapidly and succumbed to virus infection, whereas mice infected with clade 2.3.4.4f or 2.3.4.4g viruses experienced non-lethal infections. [ABSTRACT FROM AUTHOR]

On influenza virus infection or vaccination, immune responses occur, including the production of antibodies with various functions that contribute to protection from seasonal influenza virus infection. In the current study, we attempted to identify the antibody functions that play a central role in preventing the onset of seasonal influenza by comparing the levels of several antibody titers for different antibody functions between 5 subclinically infected individuals and 16 patients infected with seasonal H3N2 virus. For antibody titers before influenza virus exposure, we found that the nAb titers and enzyme-linked immunosorbent assay titers against hemagglutinin and neuraminidase (NA) proteins in the subclinically infected individuals were significantly higher than those in the patients, whereas the NA inhibition titers and antibody-dependent cellular cytotoxicity activities did not significantly differ between subclinically infected individuals and infected patients. These results suggest that nAb and enzyme-linked immunosorbent assay titers against hemagglutinin and NA serve as correlates of symptomatic influenza infection. [ABSTRACT FROM AUTHOR]

Low pathogenic H9N2 avian influenza viruses have spread in wild birds and poultry worldwide. Recently, the number of human cases of H9N2 virus infection has increased in China and other countries, heightening pandemic concerns. In Japan, H9N2 viruses are not yet enzootic; however, avian influenza viruses, including H5N1, H7N9, H5N6, and H9N2, have been repeatedly detected in raw poultry meat carried by international flight passengers from Asian countries to Japan. Although H9N2 virus-contaminated poultry products intercepted by the animal quarantine service at the Japan border have been characterized in chickens and ducks, the biological properties of those H9N2 viruses in mammals remain unclear. Here, we characterized the biological features of two H9N2 virus isolates [A/chicken/Japan/AQ-HE28-50/2016 (Ck/HE28-50) and A/chicken/Japan/AQ-HE28-57/2016 (Ck/HE28-57)] in a mouse model. We found that these H9N2 viruses replicate well in the respiratory tract of infected mice without adaptation, and that Ck/HE28-57 caused body weight loss in the infected mice. Our results indicate that H9N2 avian influenza viruses isolated from raw chicken meat products illegally brought to Japan can potentially infect and cause disease in mammals. [ABSTRACT FROM AUTHOR]

In hamsters, SARS-CoV-2 infection at the same time as or before H3N2 influenza virus infection resulted in significantly reduced influenza virus titers in the lungs and nasal turbinates. This interference may be correlated with SARS-CoV-2-induced expression of MX1. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease (COVID‐19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), was first detected in Japan in January 2020 and has spread throughout the country. Previous studies have reported that viral interference among influenza virus, rhinovirus, and other respiratory viruses can affect viral infections at the host and population level. Methods: To investigate the impact of COVID‐19 on influenza and other respiratory virus infections, we analyzed clinical specimens collected from 2244 patients in Japan with respiratory diseases between January 2018 and September 2020. Results: The frequency of influenza and other respiratory viruses (coxsackievirus A and B; echovirus; enterovirus; human coronavirus 229E, HKU1, NL63, and OC43; human metapneumovirus; human parainfluenza virus 1, 2, 3, and 4; human parechovirus; human respiratory syncytial virus; human adenovirus; human bocavirus; human parvovirus B19; herpes simplex virus type 1; and varicella‐zoster virus) was appreciably reduced among all patients during the COVID‐19 pandemic except for that of rhinovirus in children younger than 10 years, which was appreciably increased. COVID‐19 has not spread among this age group, suggesting an increased risk of rhinovirus infection in children. Conclusions: Rhinovirus infections should be continuously monitored to understand their increased risk during the COVID‐19 pandemic and viral interference with SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules. Here, the authors report bi-functional, wide tropic macrocycles that bind the influenza viral envelope protein hemagglutinin and inhibit virus infection by blocking adsorption and fusion and show efficacy in preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. [ABSTRACT FROM AUTHOR]

The avian influenza A(H7N9) virus has caused high mortality rates in humans, especially in the elderly; however, little is known about the mechanistic basis for this. In the current study, we used nonhuman primates to evaluate the effect of aging on the pathogenicity of A(H7N9) virus. We observed that A(H7N9) virus infection of aged animals (defined as age 20-26 years) caused more severe symptoms than infection of young animals (defined as age 2-3 years). In aged animals, lung inflammation was weak and virus infection was sustained. Although cytokine and chemokine expression in the lungs of most aged animals was lower than that in the lungs of young animals, 1 aged animal showed severe symptoms and dysregulated proinflammatory cytokine and chemokine production. These results suggest that attenuated or dysregulated immune responses in aged animals are responsible for the severe symptoms observed among elderly patients infected with A(H7N9) virus. [ABSTRACT FROM AUTHOR]

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus. Here, Kuroda et al. screen a library of nearly 400 interferon-stimulated genes (ISGs) and identify several ISGs that inhibit Ebola virus entry, viral transcription/replication, or virion formation. The study provides insights into interactions between Ebola and the host cells. [ABSTRACT FROM AUTHOR]

Exosomes, the extracellular vesicles that contain functional proteins and RNAs, regulate cell-cell communication. Recently, our group reported that levels of various microRNAs (miRNAs) in bronchoalveolar lavage fluid exosomes were highly increased in influenza virus-infected mice and that one of those miRNAs, miR-483-3p, was involved in the potentiation of the innate immune responses to influenza virus infection in mouse type II pneumocytes. Here, we evaluated exosomal miR-483-3p levels in the serum of influenza virus-infected mice and found that miR-483-3p levels were significantly increased during infection with a highly pathogenic avian H5N1 influenza virus. Moreover, miR-483-3p-enriched exosomes derived from type II pneumocytes potentiated the expression of proinflammatory cytokine genes in vascular endothelial cells. Our findings suggest that serum exosomal transfer of miR-483-3p might be involved in the inflammatory pathogenesis of H5N1 influenza virus infection. [ABSTRACT FROM AUTHOR]

The relationship between humans and viruses has a long history. Since the first identification of viruses in the 19th century, we have considered them to be 'pathogens' and have studied their mechanisms of replication and pathogenicity to combat the diseases that they cause. However, the relationships between hosts and viruses are various and virus infections do not necessarily cause diseases in their hosts. Rather, recent studies have shown that viral infections sometimes have beneficial effects on the biological functions and/or evolution of hosts. Here, we provide some insight into the positive side of viruses. [ABSTRACT FROM AUTHOR]

Epidemics of seasonal influenza caused by H1N1pdm09, H3N2, and type B viruses occur throughout the world. Sporadic human H5 and H7N9 virus infections are also reported in particular regions. To treat influenza patients effectively with antivirals, sensitive and broad-reactive influenza rapid diagnostic tests (IRDTs) are required. Here, we tested the sensitivity of 23 IRDTs during the 2018–2019 influenza season for their ability to detect H1N1pdm09, H3N2, H5N1, H5N6, H7N9, and Victoria- and Yamagata-lineage type B viruses. All IRDTs detected all influenza A and B viruses tested but with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar sensitivity. Such IRDTs might be useful for diagnosing patients infected with H5 and H7 viruses. [ABSTRACT FROM AUTHOR]

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine. [ABSTRACT FROM AUTHOR]

Influenza A viral ribonucleoprotein (vRNP) complexes comprise the eight genomic negative-sense RNAs, each of which is bound to multiple copies of the vRNP and a trimeric viral polymerase complex. The influenza virus life cycle centres on the vRNPs, which in turn rely on host cellular processes to carry out functions that are necessary for the successful completion of the virus life cycle. In this Review, we discuss our current knowledge about vRNP trafficking within host cells and the function of these complexes in the context of the virus life cycle, highlighting how structure contributes to function and the crucial interactions with host cell pathways, as well as on the information gaps that remain. An improved understanding of how vRNPs use host cell pathways is essential to identify mechanisms of virus pathogenicity, host adaptation and, ultimately, new targets for antiviral intervention. [ABSTRACT FROM AUTHOR]

Influenza viruses cause acute respiratory inflammation in humans and symptoms such as high fever, body aches, and fatigue. Usually these symptoms improve after several days; however, the 2009 pandemic H1N1 influenza virus [influenza A(H1N1) 2009] is more pathogenic than seasonal influenza viruses and the pathogenicity of highly pathogenic H5N1 viruses is still higher. The 1918 influenza pandemic virus caused severe pneumonia, resulting in an estimated 50 million deaths worldwide. Several virulence factors have been identified in these virus strains, but host factors are also responsible for the pathogenesis of infections caused by virulent viruses. Here, we review the contributions of both virus and host factors to the pathogenesis of these viral infections. [ABSTRACT FROM AUTHOR]

The ''Spanish'' pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/ 33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included ''Spanish Flu''- like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency. [ABSTRACT FROM AUTHOR]

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG+ memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains. [ABSTRACT FROM AUTHOR]

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. A prime example is the recent emergence of swine-origin H1N1 viruses that have transmitted to and spread among humans, resulting in outbreaks internationally. Efforts to control these outbreaks and real-time monitoring of the evolution of this virus should provide us with invaluable information to direct infectious disease control programmes and to improve understanding of the factors that determine viral pathogenicity and/or transmissibility. [ABSTRACT FROM AUTHOR]

The article presents a study that determines the binding properties of GP1 protein of Borna disease virus (BDV). Researchers carried the wild-type GP1 and a variety of GP1 deletion mutants that were FLAG-tagged at the C-terminus in human embryonic kidney 293T cells. They then purified these proteins using an anti-FLAG antibody and evaluated their ability to bind to cell lines. This study showed that GP1 bound to BDV-permissive cells but not to non-permissive cells. This protein also inhibited BDV infection via its binding to cells.

Ebola virus (EBOV), a public health concern in Africa and a potential biological weapon, is classified as a biosafety level-4 agent because of its high mortality rate and the lack of approved vaccines and antivirals. Basic research into the mechanisms of EBOV pathogenicity and the development of effective countermeasures are restricted by the current biosafety classification of EBOVs. We therefore developed biologically contained EBOV that express a reporter gene instead of the VP30 gene, which encodes an essential transcription factor. A Vero cell line that stably expresses VP30 provides this essential protein in trans and biologically confines the virus to its complete replication cycle in this cell line. This complementation approach is highly efficient because biologically contained EBOVs lacking the VP30 gene grow to titers similar to those obtained with wild-type virus. Moreover, EBOVs lacking the VP30 gene are indistinguishable in their morphology from wild-type virus and are genetically stable, as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells. We propose that this system provides a safe means to handle EBOV outside a biosafety level-4 facility and will stimulate critical studies on the EBOV life cycle as well as large-scale screening efforts for compounds with activity against this lethal virus. [ABSTRACT FROM AUTHOR]

The roles of Ebola virus (EBOV) VP24 in nucleocapsid (NC) formation and the effect of VP24 on transcription and replication of the viral genome during NC formation remain unknown. We therefore examined the effect of VP24 on the expression of a reporter gene (luciferase), viral RNA, and messenger RNA from the EBOV minigenome. VP24 inhibited the expression of luciferase and both RNAs in a dose-dependent manner, suggesting that VP24 inhibits transcription and replication of the EBOV genome. By contrast, FLAG-tagged VP24, which cannot support NC-like structure formation, did not appreciably decrease luciferase expression, indicating that association of VP24 with the ribonucleoprotein complex is required for inhibition. Glycoprotein and VP40 did not affect VP24-mediated inhibition of transcription and replication. Together, these results suggest that VP24 reduces transcription and replication of the EBOV genome by direct association with the ribonucleoprotein complex in virus-infected cells. [ABSTRACT FROM AUTHOR]

We have shown that antibody-dependent enhancement (ADE) of infection with Zaire Ebola virus (ZEBOV) is mediated by interaction of virus-specific antibodies with Fc receptors or complement component C1q and its receptors in vitro. ADE activities of the antisera to the viral glycoprotein (GP) were virus species specific and were primarily correlated with immunoglobulin (Ig) G2a and IgM levels but not with IgG1 levels. Interestingly, compared with ZEBOV, Reston Ebola virus (REBOV) had substantially weaker potential to induce ADE antibodies. Using monoclonal antibodies, we identified ZEBOV-specific ADE epitopes. To confirm epitope specificity, we constructed a chimeric ZEBOV GP, the ADE epitopes of which were replaced with the corresponding regions of REBOV GP. We found that mouse antisera to the chimeric ZEBOV GP showed less potential to induce ADE activity than did mouse antisera to wild-type ZEBOV GP, although they retained neutralizing activity. These data suggest that GP lacking the ADE-inducing epitopes may crease the potential of GP as a vaccine antigen. [ABSTRACT FROM AUTHOR]

Background. Recently, many Japanese physicians have claimed that oseltamivir is less effective in children with influenza B virus infection. This study assesses the effectiveness of oseltamivir against influenza A (H3N2) and influenza B in children on the basis of the duration of febrile illness. Methods. We used oseltamivir to treat 127 children with influenza A (H3N2; mean age, 6.97 years [range, 1–15 years]) and 362 children with influenza B (mean age, 5.16 years [range, 1–15 years]) in outpatient clinics. The duration of fever after the start of oseltamivir therapy was compared in the influenza A group and the influenza B group. Results. The mean duration of fever after the start of oseltamivir therapy was significantly greater in the influenza B group than in the influenza A (H3N2) group (2.18 days vs. 1.31 days, respectively; P<.001 ). The difference was marked in young children (1-5 years old; 2.37 days for the influenza B group vs. 1.42 days for the influenza A group) but was not significant among older children (11–15 years old). The 50% inhibitory concentration of oseltamivir against influenza B virus was nmol/L and was substantially higher than that for 75.4±41.7 type A (H3N2) virus ( nmol/L). Only 3 (1.6%) of 192 influenza B viruses were resistant to oseltamivir. 0.3±0.1 Conclusions. Oseltamivir is much less effective against influenza B virus infection in young children, probably because of the low sensitivity of influenza B viruses to oseltamivir. The effectiveness of oseltamivir against influenza B is influenced by age and host immunity. A few oseltamivir-resistant influenza B strains were isolated before the start of oseltamivir therapy. [ABSTRACT FROM AUTHOR]

The generation of vaccines for highly pathogenic avian influenza viruses, including those of the H5N1 subtype, relies on reverse genetics, which allows the production of influenza viruses from cloned cDNA. In the future, reverse genetics will likely be the method of choice for the generation of conventional influenza vaccine strains because gene reassortment by more traditional methods is cumbersome. Established systems for the artificial generation of influenza A viruses require transfection of cells with the eight to 12 plasmids that provide the eight influenza viral RNAs as well as the polymerase and nucleoproteins of the virus. However, cell lines appropriate for human vaccine production (e.g., Vero cells) cannot be transfected with high efficiencies. To overcome these problems, we established a reverse genetics system in which the eight RNA polymerase I transcription cassettes for viral RNA synthesis are combined on one plasmid. Similarly, two cassettes encoding the hemagglutinin and neuraminidase segments and six cassettes encoding the remaining proteins were combined. We also combined three RNA polymerase II transcription cassettes for the expression of the polymerase subunits. By combining these cassettes, we reduced the number of plasmids required for virus generation significantly and produced influenza A virus in Vero cells with higher efficiency than with the traditional 12 plasmid system. This new system is thus suitable for influenza virus vaccine production and may be applicable to other reverse genetics systems that rely on the introduction of several plasmids into eukaryotic cells. [ABSTRACT FROM AUTHOR]

Recent outbreaks of highly pathogenic avian influenza A virus infections (H5 and H7 subtypes) in poultry and in humans (through direct contact with infected birds) have had important economic repercussions and have raised concerns that a new influenza pandemic will occur in the near future. The eradication of pathogenic avian influenza viruses seems to be the most effective way to prevent influenza pandemics, although this strategy has not proven successful so far. Here, we review the molecular factors that contribute to the emergence of pandemic strains. [ABSTRACT FROM AUTHOR]

Viral neuraminidase inhibitors show limited efficacy in mice infected with H7N9 influenza A viruses isolated from humans. Although baloxavir marboxil protected mice from lethal challenge infection with a low pathogenic avian influenza H7N9 virus isolated from a human, its efficacy in mice infected with a recent highly pathogenic version of H7N9 human isolates is unknown. Here, we examined the efficacy of baloxavir marboxil in mice infected with a highly pathogenic human H7N9 virus, A/Guangdong/17SF003/2016. Treatment of infected mice with a single 1.5 mg/kg dose of baloxavir marboxil protected mice from the highly pathogenic human H7N9 virus infection as effectively as oseltamivir treatment at 50 mg/kg twice a day for five days. Daily treatment for five days at 15 or 50 mg/kg of baloxavir marboxil showed superior therapeutic efficacy, largely preventing virus replication in respiratory organs. These results indicate that baloxavir marboxil is a valuable candidate treatment for human patients suffering from highly pathogenic H7N9 virus infection. [ABSTRACT FROM AUTHOR]

The article discusses the recent advances and future challenges in dealing with filoviruses, previewing the papers published within the issue. According to the authors, the fact that Marburg and Ebola viruses are highly pathogenic for humans and nonhuman primates makes these viruses feared pathogens worldwide today. They note that the collection of papers in within the issue focuses on these viruses. Among others, the papers bring up to date information on the mechanisms of how filoviruses emerge and re-emerge, and the molecular mechanisms of how these viruses replicate and cause disease.

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug. [ABSTRACT FROM AUTHOR]

Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children. [ABSTRACT FROM AUTHOR]

Avian influenza viruses rarely infect humans, but the recently emerged avian H7N9 influenza viruses have caused sporadic infections in humans in China, resulting in 440 confirmed cases with 122 fatalities as of 16 May 2014. In addition, epidemiologic surveys suggest that there have been asymptomatic or mild human infections with H7N9 viruses. These viruses replicate efficiently in mammals, show limited transmissibility in ferrets and guinea pigs, and possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. In this review, we summarize the characteristic features of the novel H7N9 viruses and assess their pandemic potential. [ABSTRACT FROM AUTHOR]

Abstract: Influenza infection of humans remains an important public health problem. Vaccine strategies result in a significant but only partial control (65–85%) of infection. Thus, chemotherapeutic approaches are needed to provide a solution both for vaccine failures and to limit infection in the unvaccinated population. Previously (Walsh et al., 2011; Teijaro et al., 2011) documented that sphingosine-1-phosphate 1 receptor (S1P1R) agonists significantly protected mice against pathogenic H1N1 influenza virus by limiting immunopathologic damage while allowing host control of the infection. Here we extend that observation by documenting S1P1R agonist can control pathogenic H1N1 influenza infection in ferrets. S1P1R agonist was more effective in reducing pulmonary injury than the antiviral drug oseltamivir but, importantly, combined therapy was significantly more effective than either therapy alone. [Copyright &y& Elsevier]

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway. [ABSTRACT FROM AUTHOR]

Abstract: Real-time RT-PCR is used to quantify individual influenza viral RNAs. However, conventional real-time RT-PCR, using strand-specific primers, has been shown to produce not only the anticipated strand-specific products, but also substantial amounts of non-strand-specific products, indicating lack of specificity. Therefore, in this study, a novel strand-specific real-time RT-PCR method was established to quantify the three types of influenza viral RNA (vRNA, cRNA, and mRNA) separately. This method is based on reverse transcription using tagged primers to add a ‘tag’ sequence at the 5′ end and the hot-start method. Real-time PCR using the ‘tag’ portion as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the three types of RNA. Using this method, specific target RNA was detected at 100–100,000-folds higher level than other types of RNA. This method was also used to evaluate the vRNA, cRNA, and mRNA levels of segments 5 and 6 in MDCK cells infected with influenza A virus at different time point post-infections. The cRNA level was 1/10 to 1/100 lower than that of the vRNA and mRNA. Moreover, different dynamics of vRNA, cRNA, and mRNA synthesis were observed; the copy number of the vRNA gradually increased throughout the infection, the cRNA increased and then plateaued, while the mRNA increased and then decreased. This novel method thus provides data critical for understanding the influenza virus life cycle, including transcription, replication, and genome incorporation into virions. [Copyright &y& Elsevier]

Abstract: One year after the emergence of the 2009 influenza (H1N1) pandemic, most cases have been relatively mild; however, the possibility of enhanced viral growth ability by reassortment with seasonal viruses cannot be overlooked. Here, we show that reassortant viruses containing a seasonal H1 HA and swine-origin NA and M genes have enhanced virus growth over their wild-type parental viruses. The emergence of such viruses in nature could, therefore, represent a threat. [Copyright &y& Elsevier]

Abstract: A novel influenza (H1N1) virus caused an influenza pandemic in 2009, while highly pathogenic H5N1 avian influenza viruses have continued to infect humans since 1997. Influenza, therefore, remains a serious health threat. Currently, neuraminidase (NA) inhibitors are the mainstay for influenza therapy; however, drug-resistant mutants of seasonal H1N1 and H5N1 viruses have emerged highlighting the need for alternative therapeutic approaches. One such approach is antibody immunotherapy. Here, we show that the monoclonal antibody C179, which recognizes a neutralizing epitope common among H1, H2, H5, and H6 hemagglutinins (HAs), protected mice from a lethal challenge with various H5N1 and pandemic (H1N1) 2009 viruses when administered either intraperitoneally or intranasally. The protective efficacy of intranasally inoculated C179 was comparable to that of intraperitoneal administration. Our results suggest that direct administration of this anti-influenza antibody to viral replication sites is an effective strategy for prophylaxis and therapy. [ABSTRACT FROM AUTHOR]

Viral infections induce signaling pathways in mammalian cells that stimulate innate immune responses and affect cellular processes, such as apoptosis, mitosis, and differentiation. Here, we report that the ribosomal protein S6 kinase alpha 3 (RSK2), which is activated through the "classical" mitogen-activated protein kinase pathway, plays a role in innate immune responses to influenza virus infection. RSK2 functions in the regulation of cell growth and differentiation but was not known to play a role in the cellular antiviral response. We have found that knockdown of RSK2 enhanced viral polymerase activity and growth of influenza viruses. Influenza virus infection stimulates NK-κB- and beta interferon-dependent promoters. This stimulation was reduced in RSK2 knockdown cells, suggesting that RSK2 executes its effect through innate immune response pathways. Furthermore, RSK2 knockdown suppressed influenza virus-induced phosphorylation of the double-stranded RNA-activated protein kinase PKR, a known antiviral protein. These findings establish a role for RSK2 in the cellular antiviral response. [ABSTRACT FROM AUTHOR]

Abstract: Reassortants between type A and B influenza viruses have not been detected in nature, although both viruses co-circulate in human populations. One explanation for this may be functional incompatibility of RNA transcription and replication between type A and B viruses. To test this possibility, we constructed type A/B mosaic polymerase machinery, containing PB2, PB1, PA and nucleoprotein from each of the two virus types, and assessed their polymerase activities with a type A promoter in a reporter assay. Type B polymerase machinery containing homologous components was functional with the type A promoter albeit to various extents depending on the segments from which the regions downstream of the promoter sequence were derived, indicating functional compatibility between the type A promoter and B polymerase machinery. However, all of the A/B mosaic polymerase machinery, except that containing PA from a type A and the others from a type B virus strain, did not function with the type A promoter, indicating limited compatibility among polymerase components of both types. Taken together, these data suggest that incompatibility among components of the polymerase machinery for RNA transcription and replication alone is not responsible for the lack of heterotypic reassortants. [Copyright &y& Elsevier]

Summary: In order to prepare for the emergence of pandemic influenza viruses, we have established an influenza virus library that contains non-pathogenic influenza A virus strains with 135 combinations of 15 hemagglutinin and 9 neuraminidase subtypes. In this study, we developed a vaccine against H5N1 highly pathogenic avian influenza (HPAI) virus infection in humans using a virus strain selected from the library. We examined its immunogenic potency using cynomolgus macaques as a primate model. Virus antigen-specific antibodies were elicited by intranasal or subcutaneous administration of inactivated whole virus particle vaccines. After challenge with an H5N1 HPAI virus isolate obtained from a Vietnamese patient, the virus was detected only on next day following inoculation in the nasal and/or tracheal swabs of vaccinated macaques that were asymptomatic. On the other hand, the viruses were isolated from nasal and tracheal swabs from non-vaccinated macaques until day 5 and day 7 after inoculation of the H5N1 HPAI virus, respectively. Although six non-vaccinated macaques developed a high body temperature, and two of them lost their appetite after HPAI virus infection, they recovered by the end of the 12-day observation period and did not show the severe symptoms that have been reported in human H5N1 virus infection cases. This demonstrates that the vaccine prepared with the non-pathogenic H5N1 virus from our influenza virus library conferred protective immunity against H5N1 HPAI virus infection to macaques. [Copyright &y& Elsevier]

Abstract: Seed viruses used to produce inactivated H5N1 influenza vaccines are recombinant viruses with modified avirulent-type hemagglutinin (HA) and intact neuraminidase (NA) genes, both derived from an H5N1 isolate, and all remaining genes from the PR8 strain, which grows well in eggs. However, some reassortants grow suboptimally in eggs, imposing obstacles to timely, cost-efficient vaccine production. Here, we demonstrate that our PR8 strain supports better in ovo growth than the PR8 strain used for the WHO-recommended seed virus, NIBRG-14. Moreover, inclusion of an alternative NA protein further enhanced viral growth in eggs. These findings suggest that our H5N1 vaccine candidates would increase the availability of H5N1 vaccine doses at the onset of a new pandemic. [Copyright &y& Elsevier]

Abstract: Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract. [Copyright &y& Elsevier]

Abstract: Ebola virus causes lethal hemorrhagic fever in humans and nonhuman primates, but no effective antiviral compounds are available for the treatment of this infection. The surface glycoprotein (GP) of Ebola virus is an important target of neutralizing antibodies. Although passive transfer of GP-specific antibodies has been evaluated in mouse and guinea pig models, protection was achieved only by treatment shortly before or after virus challenge. Using these animal models, we evaluated the protective efficacy of two monoclonal antibodies whose epitopes are distinct from those of the antibodies tested by others. Treatment of mice with these antibodies 2 days after challenge completely protected most of the animals; even treatment 3 or 4 days after challenge was partially effective. Although antibody treatment in the guinea pig model was not as effective as in the mouse model, single-dose treatment of guinea pigs 1 day before, or 1 or 2 days after challenge did protect some animals. Interestingly, the protective effects seen in these animal models did not correlate with the in vitro neutralizing activity of the antibodies, suggesting different mechanisms of the neutralization by these antibodies. These results underscore the potential therapeutic utility of monoclonal antibodies for postexposure treatment of Ebola virus infections. [Copyright &y& Elsevier]

Abstract: The pandemic threat posed by highly pathogenic H5N1 influenza A viruses has created an urgent need for vaccines to protect against H5 virus infection. Because pathogenic viruses grow poorly in chicken eggs and their virulence poses a biohazard to vaccine producers, avirulent viruses produced by reverse genetics have become the preferred basis for vaccine production. Here, we investigated two key characteristics of potential H5 vaccine candidates: the hemaggutinin (HA) cleavage site sequence and its modification to attenuate virulence and the choice of background virus to provide a high-growth rate. We produced recombinant (6:2 reassortant) viruses that possessed a series of modified avirulent-type HA and neuraminidase genes, both of which were derived from an H5N1 human isolate. The other genes of these recombinant viruses were derived from donor virus strains known to grow well in eggs: the human strain A/Puerto Rico/8/34 (PR8) or an avian strain. All of the recombinant viruses grew well in eggs, were avirulent in chicks, and protected animals against infection with a wild-type virus. However, one of the recombinant viruses with an avian virus background acquired a mutation in the HA cleavage site sequence that conferred virulence potential to this virus. Moreover, vaccine candidates with the avian virus background were more virulent than those with the human virus background. We conclude that 6:2 recombinant viruses with a PR8 background are more suitable than those with an avian virus background for vaccine development and that the HA cleavage site sequence must be modified to minimize the potential for a vaccine virus to convert to a virulent form. [Copyright &y& Elsevier]

Abstract: An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed > 97% nucleotide identity with their counterparts from recent H5N1 viruses, but that the remaining two genes (PB2 and NP) were derived from other unknown viruses. This duck meat isolate was highly pathogenic to chickens upon intravenous or intranasal inoculation, replicated well in the lungs of mice and spread to the brain, but was not as pathogenic in mice as H5N1 human isolates (with a dose lethal to 50% of mice (MLD50) = 5 × 106 50% egg infectious doses [EID50]). However, viruses isolated from the brain of mice previously infected with the virus were substantially more pathogenic (MLD50 = ∼102 EID50) and possessed some amino acid substitutions relative to the original virus. These results show that poultry products contaminated with influenza viruses of high pathogenic potential to mammals are a threat to public health even in countries where the virus is not enzootic and represent a possible source of influenza outbreaks in poultry. [Copyright &y& Elsevier]

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans. [ABSTRACT FROM AUTHOR]