Search

Here, we demonstrate that a family of proteins from archaea specifically inhibit this splicing biology of all tested members of the Orthomyxoviridae family. We show that this inhibition extends to influenza A virus, influenza B virus, and isavirus genera, while having no significant impact on the mammalian transcriptome or proteome.

The human population now has two circulating respiratory RNA viruses with high pandemic potential, namely, SARS-CoV-2 and influenza A virus. As both viruses infect the airways and can result in significant morbidity and mortality, it is imperative that we also understand the consequences of getting coinfected.

The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here, we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production, and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection, but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential., One-sentence editorial summary: A microfluidic bronchial-airway-on-a-chip lined by human bronchial-airway epithelium and pulmonary endothelium can be used to rapidly identify antiviral therapeutics and prophylactics with repurposing potential.

SUMMARYSARS-CoV-2 has been found capable of inducing prolonged pathologies collectively referred to as Long-COVID. To better understand this biology, we compared the short- and long-term systemic responses in the golden hamster following either SARS-CoV-2 or influenza A virus (IAV) infection. While SARS-CoV-2 exceeded IAV in its capacity to cause injury to the lung and kidney, the most significant changes were observed in the olfactory bulb (OB) and olfactory epithelium (OE) where inflammation was visible beyond one month post SARS-CoV-2 infection. Despite a lack of detectable virus, OB/OE demonstrated microglial and T cell activation, proinflammatory cytokine production, and interferon responses that correlated with behavioral changes. These findings could be corroborated through sequencing of individuals who recovered from COVID-19, as sustained inflammation in OB/OE tissue remained evident months beyond disease resolution. These data highlight a molecular mechanism for persistent COVID-19 symptomology and characterize a small animal model to develop future therapeutics.

Morbidity and mortality in response to SARS-CoV-2 infection are significantly elevated in people of advanced age. To understand the underlying biology of this phenotype, we utilize the golden hamster model to compare how the innate and adaptive immune responses to SARS-CoV-2 infection differed between younger and older animals. We find that while both hamster cohorts showed similar virus kinetics in the lungs, the host response in older animals was dampened, with diminished tissue repair in the respiratory tract post-infection. Characterization of the adaptive immune response also revealed age-related differences, including fewer germinal center B cells in older hamsters, resulting in reduced potency of neutralizing antibodies. Moreover, older animals demonstrate elevated suppressor T cells and neutrophils in the respiratory tract, correlating with an increase in TGF-β and IL-17 induction. Together, these data support that diminished immunity is one of the underlying causes of age-related morbidity.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has been found capable of inducing long term effects commonly referred to as post-acute sequelae of SARS-CoV-2 (PASC) or long COVID. To define the molecular basis of this condition, we compared the short- and long-term responses to influenza A virus and SARSCoV-2 in the golden hamster model. These data demonstrated that SARS-CoV-2 resulted in sustained changes to lung, kidney, and brain. The most significant change in response to SARS-CoV-2 was observed in the olfactory bulb, where persistent inflammation was visible beyond one month post infection. This was characterized by microglial activation, pro-inflammatory cytokine production, and a Type I interferon (IFN-I) response in the absence of detectable virus. Given the connection between olfactory bulb injury and neurological disorders, we postulate that this prolonged inflammation is an underlying cause of long COVID. Funding Information: This work was funded by generous support from the Marc Haas Foundation, the National Institutes of Health (NCI (R01CA234614) and NIAID (2R01AI107301) and NIDDK (R01DK121072 and 1RO3DK117252) to Department of Medicine, Weill Cornell Medicine (R.E.S.)), and DARPA’s PREPARE Program (HR0011-20-2-0040). The work was further funded by NINDS (NS111251, NSO86444, NSO86444S1)(V.Z., R.A.S.). Ethics Approval Statement: The Tissue Procurement Facility operates under Institutional Review Board (IRB) approved protocol and follows guidelines set by Health Insurance Portability and Accountability Act (HIPAA). Experiments using samples from human subjects were conducted in accordance with local regulations and with the approval of the IRB at the Weill Cornell Medicine. The autopsy samples are considered human tissue research and were collected under IRB protocols 20-04021814 and 19-11021069. All autopsies have consent for research use from next of kin, and these studies were determined as exempt by IRB at Weill Cornell Medicine under those protocol numbers.

SARS-CoV-2 has caused significant morbidity and mortality across the globe. As the virus spreads, new variants are arising that show enhanced capacity to bypass pre-existing immunity. To understand the memory response to SARS-CoV-2, here we monitored SARS-CoV-2-specific T and B cells in a longitudinal study of infected and recovered golden hamsters. We demonstrate that engagement of the innate immune system following SARS-CoV-2 infection was delayed but was followed by a pronounced adaptive response. Moreover, T cell adoptive transfer conferred a reduction in virus levels and rapid induction of SARS-CoV-2-specific B cells demonstrating both lymphocyte populations contributed to the overall response. Reinfection of recovered animals with the SARS-CoV-2 beta variant showed SARS-CoV-2-specific memory T and B cells could effectively control the infection through neutralizing antibodies. These data suggest that the adaptive immune response to SARS-CoV-2 is sufficient to provide protection to the host, independent of the emergence of novel variants.

The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are activein vitroand also translatein vivo. Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivir’s therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug – amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.

The capacity to edit genomes using CRISPR-Cas systems holds immense potential for countless genetic-based diseases. However, one significant impediment preventing broad therapeutic utilization is in vivo delivery. While genetic editing at a single cell level in vitro can be achieved with high efficiency, the capacity to utilize these same biologic tools in a desired tissue in vivo remains challenging. Non-integrating RNA virus-based vectors constitute efficient platforms for transgene expression and surpass several barriers to in vivo delivery. However, the broad tissue tropism of viral vectors raises the concern for off-target effects. Moreover, prolonged expression of the Cas proteins, regardless of delivery method, can accumulate aberrant RNAs leading to unwanted immunological responses. In an effort to circumvent these shortcomings, here we describe a versatile RNA virus-based technology that can achieve cell-specific activity and self-inactivation by combining host microRNA (miRNA) biology with the CRISPR-Cas12a RNA-guided nuclease. Exploiting the RNase activity of Cas12a, we generated a vector that self-inactivates upon delivery of Cas12a and an accompanying CRISPR RNA (crRNA). Furthermore, we show that maturation of the crRNA can be made dependent on cell-specific miRNAs, which confers cell-specificity. We demonstrate that this genetic editing circuit delivers diminished yet sufficient levels of Cas12a to achieve effective genome editing whilst inducing a minimal immunological response. It can also function in a cell-specific manner thereby facilitating in vivo editing and mitigating the risk of unwanted, off-target effects.

Summary N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development., Highlights • A broadly mouse-protective anti-HA stem antibody, S9-1-10/5-1, was isolated. • S9-1-10/5-1 mainly inhibited virus release rather than virus entry. • S9-1-10/5-1 tethers virions via crosslinking HA molecules between neighboring virions. Broadly reactive human monoclonal antibodies against the influenza HA stem have received attention because of their potential utility against multiple HA subtypes. Some of these antibodies inhibit virus entry and/or protect mice via antibody-dependent cellular cytotoxicity. Here, we identified a human monoclonal antibody that suppresses virus propagation in vitro and in vivo by primarily inhibiting virus particle release. This finding provides another inhibitory mechanism of action for the anti-HA stem antibodies, indicating that the anti-HA stem antibodies could be potent anti-virals due to their pluripotency.

The emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity, mortality, and societal disruption. A better understanding of virus-host interactions may potentiate therapeutic insights toward limiting this infection. Here we investigated the dynamics of the systemic response to SARS-CoV-2 in hamsters by histological analysis and transcriptional profiling. Infection resulted in consistently high levels of virus in the upper and lower respiratory tracts and sporadic occurrence in other distal tissues. A longitudinal cohort revealed a wave of inflammation, including a type I interferon (IFN-I) response, that was evident in all tissues regardless of viral presence but was insufficient to prevent disease progression. Bolstering the antiviral response with intranasal administration of recombinant IFN-I reduced viral disease, prevented transmission, and lowered inflammation in vivo. This study defines the systemic host response to SARS-CoV-2 infection and supports use of intranasal IFN-I as an effective means of early treatment., Graphical abstract, The host response to SARS-CoV-2 results in significant inflammation. To understand this biology, Hoagland et al. utilize infected hamsters to elucidate transcriptional footprints across tissues longitudinally, showing an inflammatory response beyond the site of acute replication. Local administration of IFN-I reduces virus load and improves immune infiltrate.

Summary Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19., Graphical Abstract, Highlights • SARS-CoV-2 infection induces low IFN-I and -III levels with a moderate ISG response • Strong chemokine expression is consistent across in vitro, ex vivo, and in vivo models • Low innate antiviral defenses and high pro-inflammatory cues contribute to COVID-19, In comparison to other respiratory viruses, SARS-CoV-2 infection drives a lower antiviral transcriptional response that is marked by low IFN-I and IFN-III levels and elevated chemokine expression, which could explain the pro-inflammatory disease state associated with COVID-19.

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014–2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

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.

Summary Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo , but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.

Recently, the total number of distributed generators (DGs) such as photovoltaic generation system and wind turbine generation system connected to an actual distribution network has increased drastically. The distribution network connected to many distributed generators must be operated keeping reliability of power supply, power quality, and loss minimization. In order to accomplish active distribution network operation to take advantage of many connections of DGs, a new coordinated operation of distribution system with many connections of DGs is necessary. In this paper, the authors propose a coordinated operation of distribution network system connected to many DGs by using newly proposed sectionalizing switch control, sending voltage control, and computation of available DG connection capability. In order to check the validity of the proposed coordinated operation of distribution system, numerical simulations using the proposed coordinated distribution system operation are carried out in a practical distribution network model. © 2009 Wiley Periodicals, Inc. Electr Eng Jpn, 167(3): 46–57, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.20655

The Animal Products Safety Division (APSD) of Japan was created in the Ministry of Agriculture, Forestry and Fisheries in October 2005 to strengthen the role of the Ministry in animal production food safety. The authors outline the background to the establishment of the APSD and its functions. The APSD is engaged in administration related to risk management during the production stage of terrestrial and aquatic animal products. The APSD endeavours to ensure that risk management measures are taken in accordance with the relevant laws, regulations and administrative notifications and that they are based on risk assessment and science. The APSD works in close collaboration with other national agencies, prefecture governments, foreign governments and international organisations.