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Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1–4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

We assessed serum neutralization of Omicron BA.5 in children following SARS-CoV-2 infection during the Delta or Omicron BA.1/BA.2 variant period. Convalescent BA.5 titers were higher following infections during the Omicron BA.1/BA.2 vs Delta variant period, and in vaccinated vs unvaccinated children. Titers against BA.5 did not differ by age group.We assessed serum neutralization against antigenically advanced Omicron BA.5 in children following SARS-CoV-2 infection with Delta and Omicron BA.1/2 variants. Post-infection titers to BA.5 were significantly higher for infections during the Omicron period and in vaccinated vs unvaccinated children.

This protocol describes how to process samples potentially containing influenza A virus (IAV), amplify the samples in chicken eggs or mammalian cells and identify whether and which IAV is present.

ABSTRACTWe describe an Ebolavirusminigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirusvirus replication and/or transcription. The assay is robust (Z′ factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.

Highly pathogenic H5N1 influenza viruses have become endemic in poultry populations throughout Southeast Asia and continue to infect humans with a greater than 50% case fatality rate. So far, human-to-human transmission of these viruses has been limited. Here, we discuss the molecular features of H5N1 influenza viruses that might affect their pathogenicity, and explain the current lack of efficient human-to-human transmission. Such knowledge is critical in evaluating the pandemic risk these viruses pose.

Zusammenfassung.In der vorliegenden Arbeit erfolgt eine Bestandsaufnahme über Anwendung von und Bedarf an sportpsychologischer Diagnostik der in der Praxis des deutschen Spitzensports arbeitenden Sportpsychologen. Die Stichprobe rekrutierte sich aus den in der BISp-Sportpsychologie-Expertendatenbank (www.bisp-sportpsychologie.de) gelisteten Personen. Die Datenerhebung erfolgte mittels computergestützter Telefoninterviews. Durchschnittlich arbeiten die Sportpsychologen mit 20 Sportlern und 6 Trainern. Fast 88 % der Sportpsychologen setzen in ihrer Arbeit mit Athleten Diagnostik ein. Alle Sportpsychologen, die Diagnostik durchführen, greifen auf die Explorationstechnik zurück. Zusätzlich werden standardisierte allgemeinpsychologische (75.0 %), standardisierte sportspezifische (87.5 %) und selbst entwickelte (71.9 %) Testverfahren verwendet. Ein Großteil der Befragten berichtete u. a. über einen Bedarf an sport(art)spezifischen Verfahren.

ABSTRACTDespite the success of influenza virus vaccines in reducing severe illness, their efficacy is suboptimal. We describe here the immunogenicity and protective capacity of replication-incompetent influenza virus-like particles (VLPs) which were generated entirely from cDNAs and lacked either the entire NS gene (encoding both the NS1 and NS2 protein) or only the NS2 gene. In mammalian cells infected with NS gene-deficient VLPs, the nucleoprotein, but not other viral proteins including hemagglutinin (HA) and neuraminidase (NA), was detected. In contrast, cells infected with VLPs expressing NS1 but not NS2 (NS2 knockout) expressed multiple viral proteins, including HA and NA. When challenged with lethal doses of an antigenically homologous mouse-adapted influenza virus, 94% of mice vaccinated with the NS2-knockout VLPs survived, compared with less than 10% of those given the NS-deficient VLPs. These results demonstrate the potential of replication-incompetent NS2-knockout VLPs as novel influenza vaccines and perhaps also as vectors to express genes from entirely unrelated pathogens.

ABSTRACTEbola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.

Influenza virus reverse genetics has reached a level of sophistication where one can confidently generate virus entirely from cloned DNAs. The new systems makes it feasible to study the molecular mechanisms of virus replication and pathogenicity, as well as to generate attenuated live virus vaccines, gene delivery vehicles, and possibly other RNA viruses from cloned cDNAs. During the next decade, one can anticipate the translation of influenza virus reverse genetics into biomedically relevant advances. Copyright © 2002 John Wiley & Sons, Ltd.

Reverse genetics systems, i.e., systems for the generation of virus entirely from cloned cDNA, have been established for most nonsegmented negative-sense RNA viruses. In contrast, the generation of influenza A viruses (whose genome is composed of eight segments of negative-sense RNA) was not possible until 1999, likely due to the inherent technical difficulties of providing all eight viral RNAs as well as the four viral proteins required for replication and transcription. In 1999, we (Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9345-9350) and others (Fodor et al., 1999, J. Virol. 73, 9679-9682) demonstrated the generation of influenza A virus from plasmids, relying on the cellular enzyme RNA polymerase I for the synthesis o influenza viral RNAs. In this review, we provide background on RNA polymerase I transcription and discuss its use for the generation of influenza virus from cloned cDNAs.

In 1990, Enami et al. [PNAS 87 (1990) 3802] established a method (reverse genetics) that allowed one to generate influenza virus containing a gene segment derived from cloned cDNA. Although this method contributed tremendously to our understanding of influenza pathogenesis, the requirement of helper viruses limited its use in many experimental settings. Recently, we [PNAS 96 (1999) 9345] and others [J. Virol. 73 (1999) 9679] established systems for the generation of influenza viruses entirely from cloned cDNAs. This system requires only DNA cloning and transfection techniques, and is therefore easily adaptable by laboratories working in the field of molecular biology and virology. Thus, for the first time, a system is now available that allows highly efficient generation of influenza virus without technical limitations. Using this technology, one can now dissect influenza pathogenesis, a complex biologic process, at the molecular level.

Reverse genetics of negative-sense RNA viruses, which enables one to generate virus entirely from cloned cDNA, has progressed rapidly over the past decade. However, despite the relative ease with which nonsegmented negative-sense RNA viruses can now be produced from plasmids, the ability to generate viruses with segmented genomes has lagged considerably, largely because of the inherent technical difficulties in providing all viral RNAs and proteins from cloned cDNA. A breakthrough in reverse genetics technology in the influenza virus field came in 1999, when we (Neumann et al.,1999, Proc. Natl. Acad. Sci. USA96, 9345–9350) and others (Fodor et al.,1999, J. Virol.73, 9679–9682) exploited a new approach to viral RNA production. In this review, we discuss the background for this advance, the systems that are now available for the generation of influenza viruses, and the implications of these developments for the future of virus research.

ABSTRACTThe importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimerization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.

ABSTRACTViral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.

We present a system for creating influenza virus by generating viral RNA (vRNA) and mRNA from one template. Recently, a system for the generation of influenza A virus entirely from cloned cDNAs was established (Neumann et al.,1999, Proc. Natl. Acad. Sci. USA96, 9345–9350). Cells were transfected with plasmids for RNA polymerase I-driven intracellular synthesis of all eight viral RNAs, and with protein expression plasmids for the synthesis of viral structural proteins. Although this system is highly efficient in virus generation, the construction and cotransfection of 17 plasmids is cumbersome and may limit the use of this system to cell lines that can be transfected with high efficiencies. Synthesizing both vRNA and mRNA from one template would reduce the number of plasmids required for virus generation. Therefore, we generated a bidirectional transcription construct that contains cDNA encoding PB1 flanked by an RNA polymerase I (pol I) promoter for vRNA synthesis and an RNA polymerase II (pol II) promoter for mRNA synthesis. The utility of this approach is proved by the generation of virus after transfecting the pol I/pol II-promoter–PB1 construct together with vRNA- and protein-expression constructs for the remaining seven segments. Because this approach reduces the number of plasmids required for virus generation, it also reduces the work necessary for cloning, probably enhances the efficiency of virus generation, and expands the use of the reverse-genetics system to cell lines for which efficient cotransfection of 17 plasmids cannot be achieved.

ABSTRACTWe established a plasmid-based system for generating infectious influenza virus-like particles entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with plasmids encoding the influenza A virus structural proteins and with a plasmid encoding an influenza virus-like viral RNA (vRNA) which contained an antisense copy of the cDNA for green fluorescence protein (GFP) flanked by an RNA polymerase I promoter and terminator. Intracellular transcription of the latter construct by RNA polymerase I generated GFP vRNA that was packaged into influenza virus-like particles. This system, which produced more than 104infectious particles per ml of supernatant, would be useful in studies of influenza virus replication and particle formation. It might also benefit efforts in vaccine production and in the development of improved gene therapy vectors.

The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Ensuring that COVID-19 vaccines are delivered to older populations and those living in low resource settings is essential to end the pandemic.

We established an efficient plasmid-driven system for the generation of infectious influenza VLPs containing a virus-like RNA segment entirely from cDNAs. To generate influenza VLPs, we used the RNA polymerase I system for the intracellular synthesis of influenza virus RNAs. Human embryonic kidney cells (293T) were transfected with plasmids encoding the influenza A virus structural proteins and with a plasmid encoding an influenza virus-like viral RNA (vRNA), which contained an antisense copy of the cDNA for green fluorescence protein (GFP) flanked by an RNA polymerase I promoter and terminator (pPolI-GFP). Influenza virus-like particles containing GFP vRNA that were infectious and expressed GFP in infected cells were generated. We also generated VLPs lacking the NS gene by eliminating the plasmid for the NS RNA segment from the set of plasmids required for infectious influenza virus production. Because of its efficiency, this system would be useful in studies of influenza virus replication and particle formation as well as for production of vaccines.

RNA polymerase I transcription has been used for expression of influenza vRNA molecules, with influenza hemagglutinin or other cDNAs precisely inserted between mouse rDNA promoter and terminator sequences. In in vitro studies generation of HA vRNA transcripts in high rates and correct formation of their 5' ends as well as their 3' ends has been achieved for such hybrid DNA templates. For in vivo expression studies, the HA coding region was replaced by chloramphenicol acetyltransferase (CAT), also in vRNA antisense orientation, with both influenza terminal sequences beyond start and stop codons being retained on the resulting transcript. Following transfection with precisely constructed hybrid DNA templates and depending on infection with influenza virus, CAT activity could be demonstrated. Templates resulting in 3' extended vRNA molecules did not give this result. vRNA-CAT molecules were not only recognized by influenza viral RNA polymerase for synthesis of plus strand mRNAs, but also were packaged into progeny virus particles, as shown by CAT activity in infected cells after passaging of virus containing supernatants. Copyright 1994, 1999 Academic Press

ABSTRACTThe broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications.IMPORTANCEThis work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis.

RNA polymerase I has been used for transcription of Influenza hemagglutinln (HA) cDNA precisely linked In the anti-sense configuration to both mouse rDNA promoter and terminator segments. In transcription reactions based on Ehrllch ascltes cell nuclear extracts, specific uniform RNA products are synthesized In high rates, that are comparable to original rDNA template transcriptions. Primer extension reactions show the 5′ ends of these RNA transcripts to be located exactly at position +1 , corresponding to the 5′ end of negative strand HA viral RNA. RNA 3′ ends in a first series of constructs were found extended beyond the accepted location of pre-rRNA 3′ ends, in using both hybrid cDNA and original rDNA templates. But upon deletion of six basepairs from the rDNA termination region RNA polymerase I transcription has been adapted to yield correctly terminated Influenza viral RNA in vitro. This result has been confirmed In an in vivo experiment via synthesis of an anti-sense viral RNA molecule containing the chloramphenlcol acetyltransferase (CAT) gene, which In turn is recognized at Its terminal sequence by viral RNA dependent RNA polymerase for plus strand mRNA synthesis and expression of CAT activity.