Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

Human Oral Mucosa Stem Cells Increase Survival of Neurons Affected by In Vitro Anoxia and Improve Recovery of Mice Affected by Stroke Through Time-limited Secretion of miR-514A-3p.

The success rate of regenerative medicine largely depends on the type of stem cells applied in such procedures. Consequently, to achieve the needed level for clinical standardization, we need to investigate the viability of accessible sources with sufficient quantity of cells. Since the oral region partly originates from the neural crest, which naturally develops in niche with decreased levels of oxygen, the main goal of this work was to test if human oral mucosa stem cells (hOMSC) might be used to treat neurons damaged by anoxia. Here we show that hOMSC are more resistant to anoxia than human induced pluripotent stem cells and that they secrete BDNF, GDNF, VEGF and NGF. When hOMSC were added to human neurons damaged by anoxia, they significantly improved their survival. This regenerative capability was at least partly achieved through miR-514A-3p and SHP-2 and it decreased in hOMSC exposed to neural cells for 14 or 28 days. In addition, the beneficial effect of hOMSC were also confirmed in mice affected by stroke. Hence, in this work we have confirmed that hOMSC, in a time-limited manner, improve the survival of anoxia-damaged neurons and significantly contribute to the recovery of experimental animals following stroke.

Evaluation of the Antiviral Efficacy of Subcutaneous Nafamostat Formulated with Glycyrrhizic Acid against SARS-CoV-2 in a Murine Model.

The ongoing COVID-19 pandemic highlights the urgent need for effective antiviral agents and vaccines. Drug repositioning, which involves modifying existing drugs, offers a promising approach for expediting the development of novel therapeutics. In this study, we developed a new drug, MDB-MDB-601a-NM, by modifying the existing drug nafamostat (NM) with the incorporation of glycyrrhizic acid (GA). We assessed the pharmacokinetic profiles of MDB-601a-NM and nafamostat in Sprague-Dawley rats, revealing rapid clearance of nafamostat and sustained drug concentration of MDB-601a-NM after subcutaneous administration. Single-dose toxicity studies showed potential toxicity and persistent swelling at the injection site with high-dose administration of MDB-601a-NM. Furthermore, we evaluated the efficacy of MDB-601a-NM in protecting against SARS-CoV-2 infection using the K18 hACE-2 transgenic mouse model. Mice treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM exhibited improved protectivity in terms of weight loss and survival rates compared to the nafamostat-treated group. Histopathological analysis revealed dose-dependent improvements in histopathological changes and enhanced inhibitory efficacy in MDB-601a-NM-treated groups. Notably, no viral replication was detected in the brain tissue when mice were treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM. Our developed MDB-601a-NM, a modified Nafamostat with glycyrrhizic acid, shows improved protectivity against SARS-CoV-2 infection. Its sustained drug concentration after subcutaneous administration and dose-dependent improvements makes it a promising therapeutic option.

Intrinsic variation effect in memristive neural network with weight quantization.

To analyze the effect of the intrinsic variations of the memristor device on the neuromorphic system, we fabricated 32 × 32 Al2O3/TiOx-based memristor crossbar array and implemented 3 bit multilevel conductance as weight quantization by utilizing the switching characteristics to minimize the performance degradation of the neural network. The tuning operation for 8 weight levels was confirmed with a tolerance of ±4µA (±40µS). The endurance and retention characteristics were also verified, and the random telegraph noise (RTN) characteristics were measured according to the weight range to evaluate the internal stochastic variation effect. Subsequently, a memristive neural network was constructed by off-chip training with differential memristor pairs for the Modified National Institute of Standards and Technology (MNIST) handwritten dataset. The pre-trained weights were quantized, and the classification accuracy was evaluated by applying the intrinsic variations to each quantized weight. The intrinsic variations were applied using the measured weight inaccuracy given by the tuning tolerance, RTN characteristics, and the fault device yield. We believe these results should be considered when the pre-trained weights are transferred to a memristive neural network by off-chip training.

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool.

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 104 RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

In Vitro Profiling of Laninamivir-Resistant Substitutions in N3 to N9 Avian Influenza Virus Neuraminidase Subtypes and Their Association with In Vivo Susceptibility.

Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3R292K) or Q136K (ma81K-N8Q136K) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3R292K and ma81K-N8Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs.IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP).

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection.

Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs-particularly newly introduced treatments-are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T-which is located outside of the catalytic or framework residue of the NA active site-exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading.IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform.

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor.

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

An I436N substitution confers resistance of influenza A(H1N1)pdm09 viruses to multiple neuraminidase inhibitors without affecting viral fitness.

The resistance of influenza viruses to neuraminidase (NA) inhibitors (NAIs; i.e. oseltamivir, zanamivir, peramivir and laninamivir) can be associated with several NA substitutions, with differing effects on viral fitness. To identify novel molecular markers conferring multi-NAI resistance, the NA gene of oseltamivir-resistant (H275Y, N1 numbering) 2009 pandemic influenza [A(H1N1)pdm09] virus was enriched with random mutations. This randomly mutated viral library was propagated in Madin-Darby canine kidney (MDCK) cells under zanamivir pressure and gave rise to additional changes within NA, including an I436N substitution located outside the NA enzyme active site. We generated four recombinant A(H1N1)pdm09 viruses containing either wild-type NA or NA with single (I436N or H275Y) or double (H275Y-I436N) substitutions. The double H275Y-I436N mutation significantly reduced inhibition by oseltamivir and peramivir and reduced inhibition by zanamivir and laninamivir. I436N alone reduced inhibition by all NAIs, suggesting that it is a multi-NAI resistance marker. I436N did not affect viral fitness in vitro or in a murine model; however, H275Y and I436N together had a negative impact on viral fitness. Further, I436N alone did not have an appreciable impact on viral replication in the upper respiratory tract or transmissibility in ferrets. However, the rg-H275Y-I436N double mutant transmitted less efficiently than either single mutant via the direct contact and respiratory droplet routes in ferrets. Overall, these results highlight the usefulness of a random mutagenesis approach for identifying potential molecular markers of resistance and the importance of I436N NA substitution in A(H1N1)pdm09 virus as a marker for multi-NAI resistance.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

Virological and pathological characterization of an avian H1N1 influenza A virus.

Gene segments from avian H1N1 influenza A viruses have reassorted with other influenza viruses to generate pandemic strains over the past century. Nevertheless, little effort has been invested in understanding the characteristics of avian H1N1 influenza viruses. Here, we present the genome sequence and a molecular and virological characterization of an avian influenza A virus, A/wild bird/Korea/SK14/2014 (A/SK14, H1N1), isolated from migratory birds in South Korea during the winter season of 2014-2015. Full-genome sequencing and phylogenetic analysis revealed that the virus belongs to the Eurasian avian lineage. Although it retained avian-receptor binding preference, A/SK14 virus also exhibited detectable human-like receptor binding and was able to replicate in differentiated primary normal human bronchial epithelial cells. In animal models, A/SK14 virus was moderately pathogenic in mice, and virus was detected in nasal washes from inoculated guinea pigs, but not in direct-contact guinea pigs. Although A/SK14 showed moderate pathogenicity and no evidence of transmission in a mammalian model, our results suggest that the dual receptor specificity of A/SK14-like virus might allow for a more rapid adaptation to mammals, emphasizing the importance of further continuous surveillance and risk-assessment activities.

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses.

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.

Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).

Molecular Markers for Interspecies Transmission of Avian Influenza Viruses in Mammalian Hosts.

In the last decade, a wide range of avian influenza viruses (AIVs) have infected various mammalian hosts and continuously threaten both human and animal health. It is a result of overcoming the inter-species barrier which is mostly associated with gene reassortment and accumulation of mutations in their gene segments. Several recent studies have shed insights into the phenotypic and genetic changes that are involved in the interspecies transmission of AIVs. These studies have a major focus on transmission from avian to mammalian species due to the high zoonotic potential of the viruses. As more mammalian species have been infected with these viruses, there is higher risk of genetic evolution of these viruses that may lead to the next human pandemic which represents and raises public health concern. Thus, understanding the mechanism of interspecies transmission and molecular determinants through which the emerging AIVs can acquire the ability to transmit to humans and other mammals is an important key in evaluating the potential risk caused by AIVs among humans. Here, we summarize previous and recent studies on molecular markers that are specifically involved in the transmission of avian-derived influenza viruses to various mammalian hosts including humans, pigs, horses, dogs, and marine mammals.