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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) often causes severe viral pneumonia. Although many studies using mouse models have examined the pathogenicity of SARS-CoV-2, COVID-19 pathogenesis remains poorly understood. In vivo imaging analysis using two-photon excitation microscopy (TPEM) is useful for elucidating the pathology of COVID-19, providing pathological insights that are not available from conventional histological analysis. However, there is no reporter SARS-CoV-2 that demonstrates pathogenicity in C57BL/6 mice and emits sufficient light intensity for two-photon in vivo imaging. Here, we generated a mouse-adapted strain of SARS-CoV-2 (named MASCV2-p25) and demonstrated its efficient replication in the lungs of C57BL/6 mice, causing fatal pneumonia. Histopathologic analysis revealed the severe inflammation and infiltration of immune cells in the lungs of MASCV2-p25-infected C57BL/6 mice, not unlike that observed in COVID-19 patients with severe pneumonia. Subsequently, we generated a mouse-adapted reporter SARS-CoV-2 (named MASCV-Venus-p9) by inserting the fluorescent protein-encoding gene Venus into MASCV2-p25 and sequential lung-to-lung passages in C57BL/6 mice. C57BL/6 mice infected with MASCV2-Venus-p9 exhibited severe pneumonia. In addition, the TPEM of the lungs of the infected C57BL/6J mice showed that the infected cells emitted sufficient levels of fluorescence for easy observation. These findings suggest that MASCV2-Venus-p9 will be useful for two-photon in vivo imaging studies of the pathogenesis of severe COVID-19 pneumonia. [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]

Background: Immunocompromised patients infected with influenza virus require prolonged treatment with neuraminidase inhibitors, because these patients are not able to eradicate the virus from the respiratory tract, leading to the emergence of drug-resistant mutant viruses.Methods: In this study, we examined the efficacy of baloxavir marboxil in nude mice that were immunologically deficient.Results: Daily treatment with a suboptimal dose of baloxavir marboxil increased the survival time of the virus-infected nude mice but did not clear the virus from their respiratory organs, resulting in gradual body weight loss after termination of treatment.Conclusions: Despite the prolonged baloxavir marboxil treatment, few resistant mutants were detected. [ABSTRACT FROM AUTHOR]

Prolonged treatment of immunocompromised influenza patients with viral neuraminidase (NA) inhibitors is required, because the immune system of such patients fails to eradicate the viruses. Here, we attempted to eradicate influenza virus from the respiratory organs of nude mice, which is a model of immunocompromised hosts, by using combination therapy of the viral polymerase inhibitor favipiravir and monoclonal antibodies (mAbs) against the receptor-binding site (RBS) and stem of viral hemagglutinin (HA). Although monotherapy or combination therapy of two antivirals (two mAbs or favipiravir plus a mAb) suppressed virus replication, they failed to eradicate viruses from nude mice. In contrast, the triple combination therapy of favipiravir plus anti-Stem and anti-RBS mAbs completely stopped virus replication in nude mice, resulting in virus clearance. Triple combination approaches should be considered for the treatment of human immunocompromised patients with severe influenza. [ABSTRACT FROM AUTHOR]

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global pandemic. Although several vaccines targeting SARS-CoV-2 spike proteins protect against COVID-19 infection, mutations affecting virus transmissibility and immune evasion potential have reduced their efficacy, leading to the need for a more efficient strategy. Available clinical evidence regarding COVID-19 suggests that endothelial dysfunction with thrombosis is a central pathogenesis of progression to systemic disease, in which overexpression of plasminogen activator inhibitor-1 (PAI-1) may be important. Here we developed a novel peptide vaccine against PAI-1 and evaluated its effect on lipopolysaccharide (LPS)-induced sepsis and SARS-CoV-2 infection in mice. Administration of LPS and mouse-adapted SARS-CoV-2 increased serum PAI-1 levels, although the latter showed smaller levels. In an LPS-induced sepsis model, mice immunized with PAI-1 vaccine showed reduced organ damage and microvascular thrombosis and improved survival compared with vehicle-treated mice. In plasma clot lysis assays, vaccination-induced serum IgG antibodies were fibrinolytic. However, in a SARS-CoV-2 infection model, survival and symptom severity (i.e., body weight reduction) did not differ between vaccine- and vehicle-treated groups. These results indicate that although PAI-1 may promote the severity of sepsis by increasing thrombus formation, it might not be a major contributor to COVID-19 exacerbation. [ABSTRACT FROM AUTHOR]

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential. [ABSTRACT FROM AUTHOR]

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are cur-rently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previ-ously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-express-ing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (HlNlpdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a con-ventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines. [ABSTRACT FROM AUTHOR]

Background. The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in influenza A virus subtype H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However, the in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood.Methods. We created single-point-mutant viruses with 3 genetically different backgrounds (ie, 1 belonging to clade 1 and 2 belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico.Results. The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in 2 of 3 virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir.Conclusions. Our findings provide new insight into the mechanism of NA-I117V–mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses. [ABSTRACT FROM AUTHOR]

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses. [ABSTRACT FROM AUTHOR]

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 μg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants. [ABSTRACT FROM AUTHOR]

The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivirresistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouseadapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients. [ABSTRACT FROM AUTHOR]

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 μM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19. [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]

During the Omicron wave, previous variants such as BA.2, BA.4, and BA.5 were replaced by newer variants with additional mutations in the spike protein. These variants, BA.4.6, BQ.1.1, and XBB, have spread in different countries with different degrees of success. Here, we evaluated the replicative ability and pathogenicity of BA.4.6, BQ1.1, and XBB clinical isolates in male Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with these Omicron subvariants, the replicative ability of BQ.1.1 and XBB in lung tissue was higher than that of BA.4.6 and BA.5. Of note, BQ.1.1 was lethal in both male and female transgenic human ACE2 hamsters. In competition assays, XBB replicated better than BQ.1.1 in the nasal turbinate tissues of female hamsters previously infected with Omicron BA.2. These results suggest that newer Omicron subvariants in the XBB family are still evolving and should be closely monitored. Omicron variants BQ.1.1 and XBB increased pathogenicity in wild-type hamsters, with BQ.1.1 showing higher mortality in human ACE2 transgenic hamsters compared to earlier Omicron variants.. [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]

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi‐basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ‐HE29‐22/2017 (HE29‐22) and A/duck/Japan/AQ‐HE29‐52/2017 (HE29‐52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ‐HE28‐3/2016 (HE28‐3)] in mice and ferrets. In mice, HE29‐52 was more pathogenic than HE29‐22 and HE28‐3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan. [ABSTRACT FROM AUTHOR]

An abstract of the article "Effect of an Asparagine-to-Serine Mutation at Position 294 in Neuraminidase on the Pathogenicity of Highly Pathogenic H5N1 Influenza A Virus," by Maki Kiso and colleagues is presented.

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]

Highlights: [•] PB2-KO virus bearing HN of HPIV3 did not replicate in normal cells. [•] PB2-KO virus bearing HN of HPIV3 elicited mucosal and systemic immunity. [•] Hamsters immunized with PB2-KO virus bearing HN of HPIV3 were protected from both influenza virus and parainfluenza virus challenge. [ABSTRACT FROM AUTHOR]

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic. [ABSTRACT FROM AUTHOR]

Abstract: The wide distribution of H5N1 highly pathogenic avian influenza viruses is a global threat to human health. Indonesia has had the largest number of human infections and fatalities caused by these viruses. To understand the enzootic conditions of the viruses in Indonesia, twenty-four H5N1 viruses isolated from poultry from 2003 to 2007 were phylogenetically characterized. Although previous studies exclusively classified the Indonesian viruses into clades 2.1.1–2.1.3, our phylogenetic analyses showed a new sublineage that did not belong to any of the present clades. In addition, novel reassortant viruses were identified that emerged between this new sublineage and other clades in 2005–2006 on Java Island. H5N1 viruses were introduced from Java Island to Sulawesi, Kalimantan, and Sumatra Island on multiple occasions from 2003–2007, causing the geographical expansion of these viruses in Indonesia. These findings identify Java Island as the epicenter of the Indonesian H5N1 virus expansion. [Copyright &y& Elsevier]

Highly pathogenic avian influenza H5N1 viruses are circulating in many countries. We recently discovered that these viruses have been transmitted to pigs on multiple occasions in Indonesia. To investigate whether avian H5N1 influenza viruses adapted to mammals through their introduction into pigs, we examined the growth of avian and swine isolates in cell culture and compared their pathogenicity in mice. We found that swine isolates were less virulent to mice than avian isolates, suggesting that the viruses became attenuated during their replication in pigs. Continuous surveillance of H5N1 viruses among pigs is clearly warranted. [ABSTRACT FROM AUTHOR]

To clarify the threat posed by emergence of highly pathogenic influenza A(H7N9) virus infection among humans, we characterized the viral polymerase complex. Polymerase basic 2-482R, polymerase basic 2-588V, and polymerase acidic-497R individually or additively enhanced virus polymerase activity, indicating that multiple replication-enhancing mutations in 1 isolate may contribute to virulence. [ABSTRACT FROM AUTHOR]

Background: Treatment of immunocompromised, influenza virus-infected patients with the viral neuraminidase inhibitor oseltamivir often leads to the emergence of drug-resistant variants. Combination therapy with compounds that target different steps in the viral life cycle may improve treatment outcomes and reduce the emergence of drug-resistant variants.Methods: Here, we infected immunocompromised nude mice with an influenza A virus and treated them with neuraminidase (oseltamivir, laninamivir) or viral polymerase (favipiravir) inhibitors, or combinations thereof.Results: Combination therapy for 28 days increased survival times compared with monotherapy, but the animals died after treatment was terminated. Mono- and combination therapies did not consistently reduce lung virus titers. Prolonged viral replication led to the emergence of neuraminidase inhibitor-resistant variants, although viruses remained sensitive to favipiravir. Overall, favipiravir provided greater benefit than neuraminidase inhibitors.Conclusions: Collectively, our data demonstrate that combination therapy in immunocompromised hosts increases survival times, but does not suppress the emergence of neuraminidase inhibitor-resistant variants. [ABSTRACT FROM AUTHOR]

Vaccination is the first line of protection against influenza virus infection in humans. Although inactivated and live-attenuated vaccines are available, each vaccine has drawbacks in terms of immunogenicity and safety. To overcome these issues, our group has developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2-expressing cells. Here we generated PB2-KO viruses possessing the hemagglutinin (HA) and neuraminidase (NA) segments from H1N1pdm09 or type B viruses and tested their vaccine potential. The two PB2-KO viruses propagated efficiently in PB2-expressing cells, and expressed chimeric HA as expected. Virus-specific IgG and IgA antibodies were detected in mice immunized with the viruses, and the immunized mice showed milder clinical signs and/or lower virus replication levels in the respiratory tract upon virus challenge. Our results indicate that these PB2-KO viruses have potential as vaccine candidates. [ABSTRACT FROM AUTHOR]

Background: Secondary bacterial infections after influenza can be a serious problem, especially in young children and the elderly, yet the efficacy of current vaccines is limited. Earlier work demonstrated that a replication-incompetent PB2-knockout (PB2-KO) influenza virus possessing a foreign gene in the coding region of its PB2 segment can serve as a platform for a bivalent vaccine.Methods: In the current study, we generated the PB2-KO virus expressing pneumococcal surface protein A (PspA), PB2-KO-PspA virus, the replication of which is restricted to PB2-expressing cells. We then examined the protective efficacy of intranasal immunization with this virus as a bivalent vaccine in a mouse model.Results: High levels of influenza virus-specific and PspA-specific antibodies were induced in the serum and airways of immunized mice. The intranasally immunized mice were protected from lethal doses of influenza virus or Streptococcus pneumoniae. These mice were also completely protected from secondary pneumococcal pneumonia after influenza virus infection.Conclusions: These findings indicate that our recombinant influenza virus serves as a novel and powerful bivalent vaccine against primary and secondary pneumococcal pneumonia as well as influenza. [ABSTRACT FROM AUTHOR]

Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures. [ABSTRACT FROM AUTHOR]

Although raccoons (Procyon Iotor) are susceptible to influenza viruses, highly pathogenic avian influenza virus (H5N1) infection in these animals has not been reported. We performed a serosurvey of apparently healthy feral raccoons in Japan and found specific antibodies to subtype H5N1 viruses. Feral raccoons may pose a risk to farms and public health. [ABSTRACT FROM AUTHOR]

Background. Although influenza virus resistance to the neuraminidase inhibitor zanamivir is reported less frequently than is resistance to the neuraminidase inhibitor oseltamivir in clinical settings, it is unknown whether this difference is due to the limited use of zanamivir or to an inherent property of the drug. We therefore compared the prevalence of drug-resistant viruses and virus shedding in seasonal influenza virus-infected children treated with either oseltamivir or zanamivir. Methods. Clinical specimens (throat or nasal swab) were collected from a total of 144 pediatric influenza patients during the 2005-2006, 2006-2007, 2007-2008, and 2008-2009 influenza seasons. Neuraminidase inhibitor-resistant mutants were detected among the isolated viruses by sequencing the viral hemagglutinin and neuraminidase genes. Sensitivity of the viruses to neuraminidase inhibitors was tested by neuraminidase inhibition assay. Results. In oseltamivir- or zanamivir-treated influenza patients who were statistically comparable in their age distribution, vaccination history, and type or subtype of virus isolates, the virus-shedding period in zanamivirtreated patients was significantly shorter than that in oseltamivir-treated patients. Furthermore, the frequency of zanamivir-resistant viruses was significantly lower than that of oseltamivir-resistant viruses. Conclusion. In comparison with treatment with oseltamivir, treatment of pediatric patients with zanamivir resulted in the emergence of fewer drug-resistant influenza viruses and a shorter virus-shedding period. We conclude that zanamivir shows promise as a better therapy for pediatric influenza patients. [ABSTRACT FROM AUTHOR]

Although two classes of antiviral drugs, M2 ion channel blockers and neuraminidase (NA) inhibitors, are available to combat seasonal H1N1 and H3N2, and highly pathogenic avian (e.g., H5N1) influenza, the emergence and spread of drug-resistant viruses is of great concern. Animal studies suggest that combination chemotherapy with M2 and NA inhibitors may be an effective option to reduce the emergence of drug-resistant viruses. In this study, we evaluated the antiviral susceptibility of clinical isolates from immunocompetent children infected with a seasonal influenza A virus and treated with a combination of amantadine and oseltamivir. We found that amantadine-resistant viruses emerged during this combination treatment. While viruses with mutations known to confer oseltamivir resistance were not detected, we found viruses with mutations in NA that reduced sialidase activity and viruses with hemagglutinin mutations. These findings suggest that while the amantadine-oseltamivir combination is an effective therapeutic option for influenza, drug-resistant viruses do appear with this combination therapy. [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]

Abstract: H5N1 highly pathogenic avian influenza viruses evolved into several clades, leading to appreciably distinct antigenicities of their hemagglutinins. As such, candidate H5N1 pre-pandemic vaccines for human use should be sought. Here, to evaluate fundamental immunogenic variations between H5N1 vaccines, we prepared four inactivated H5N1 test vaccines from different phylogenetic clades (clade 1, 2.1, 2.2, and 2.3.4) in accordance with the WHO recommendation, and tested their cross-clade immunity in a mouse model by vaccination followed by challenge with heterologous virulent viruses. All H5N1 vaccines tested provided full or partial cross-clade protective immunity, except one clade 2.2-based vaccine, which did not protect mice from clade 2.3.4 virus challenge. Among the test vaccines, a clade 2.1-based vaccine possessed the broadest-spectrum cross-immunity. These results suggest that currently stockpiled pre-pandemic vaccines, especially clade 2.1-based vaccines, will likely be useful as backup vaccines in a pandemic situation, even one involving antigenic-drifted viruses. [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]

The article presents a medical research study that examined the prevalence and transmission of influenza B viruses with reduced sensitivity to neuraminidase inhibitors. Influenza B isolates from children before and after oseltamivir therapy and from untreated influenza patients in Japan. The drug sensitivities of the influenza B viruses were tested through sialidase inhibition assay. The authors conclude that influenza B viruses with reduced neuraminidase inhibitor sensitivity did not occur as frequently as resistant influenza A viruses. The viruses are transmitted within communities and families.

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]

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-α2,6-galactose (SAα2,6Gal), whereas the latter prefer those ending in SAα2,3Gal (refs 3–6). A conversion from SAα2,3Gal to SAα2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates. [ABSTRACT FROM AUTHOR]

Abstract: In Japan, between the end of December 2003 and March 2004, four outbreaks of acute, highly transmissible and lethal disease occurred in birds in three prefectures separated by 150–450 km, involving three chicken farms and a group of chickens raised as pets. The cause of each outbreak was an H5N1 influenza A virus—the first highly pathogenic virus to be isolated from the outbreaks in Japan since 1925. The H5N1 virus was also isolated from dead crows, apparently infected by contact with virus-contaminated material. These H5N1 viruses were antigenically similar to each other, but could be differentiated from other H5 viruses, including those isolated from Hong Kong in 1997 and 2003, by use of a panel of monoclonal antibodies in hemagglutination inhibition assays. Genetically, the H5N1 viruses in Japan were closely related to each other in all genes and were genetically closely related to a single isolate of genotype V that was isolated in 2003 in the Guandong Province of mainland China (A/chicken/Shantou/4231/2003). The virulence of the index isolate (A/chicken/Yamaguchi/7/2004) was studied in chickens and mice. Chickens intravenously or intranasally inoculated with the isolate died within 1 or 3 days of inoculation, respectively. In mice, although this virus replicated well in the lung without prior adaptation and spread to the brain, the dose lethal to 50% of the mice was 5 × 105 50% egg infectious doses (EID50), which is less pathogenic than the Hong Kong 1997 H5N1 viruses isolated from humans. Our findings indicate that the H5N1 viruses associated with the influenza outbreaks in chickens in Japan were genotypically closely related to an H5N1 virus isolated from chicken in China in 2003 (genotype V), but were different from those prevalent in southeastern Asia in 2003–2004 (i.e., genotype Z) and that these highly pathogenic viruses can be transmitted to crows, which are highly susceptible to these viruses. [Copyright &y& Elsevier]

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA- 222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA- 222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA- 222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans. [ABSTRACT FROM AUTHOR]

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wildtype virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxyterminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis. [ABSTRACT FROM AUTHOR]