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Ausbrüche neuartiger oder bei uns sonst nicht heimischer, sich aber stetig ausbreitender Erreger sind ein fester Bestandteil unserer globalisierten und eng vernetzten Welt geworden. Nach der SARS-CoV-2-Pandemie haben die Affenpocken 2022 bewiesen, dass sich nicht nur neuartige, agile, kleine RNA-Viren in Windeseile auf dem gesamten Erdball ausbreiten können, sondern auch eher konservative DNA-Viren, denen man bisher ein sehr festes Verbreitungsgebiet zugeordnet hatte. Die Palette an Erregern, die vom endemischen zum epi- oder pandemischen Ausbruchsgeschehen führen und schwerwiegende Krankheitsbilder verursachen können, ist groß und steigt durch die ständige Entstehung neuartiger Krankheitserreger noch weiter an. Die Überwachung des weltweiten Infektionsgeschehens und die Entwicklung und Anpassung zeitgemäßer Diagnostikverfahren als Vorbereitung oder rasche Antwort auf Ausbruchsgeschehen kann von der Routinediagnostik nicht geleistet werden, sondern ist Aufgabe spezialisierter Nischeninstitute mit besonderem Auftrag, wie es das Institut für Mikrobiologie der Bundeswehr (IMB) als Ressortforschungseinrichtung des Bundes ist. Nachfolgend wird dargestellt, wie das IMB auf den Affenpockenausbruch vorbereitet war und wie die Diagnostik bedarfsorientiert abläuft., Novel and emerging viruses have integrated themselves firmly into our globalized world. After the SARSCoV-2 pandemic monkeypox virus has proven that not only novel and quickly mutating RNA viruses can rapidly spread around the globe and cause pandemics but also a rather conservative DNA virus that was thought to have a relatively small and fix area of distribution. The panel of highly pathogenic microorganisms that have the potential to turn from locally endemic to epidemic or pandemic agents is quite extensive and even larger when the constant evolution of novel novel microorganisms is taken into account. Only highly specialized institutions with a distinct mission like the Bundeswehr Institute of Microbiology (IMB) can tackle the broad spectrum of constant monitoring of infectious disease processes worldwide and the development of state-of-the-art diagnostics for expected and unexpected pathogens of high consequnece. The following essay is a short description of the preparedness and activities in poxviral diagnostics and research of the IMB and an outlook on future activities.

Background: In coronavirus disease 2019 (COVID-19), current case definitions presume mainly lower respiratory tract infection. However, cases seen outside the epicenter of the epidemic may differ in their overall clinical appearance due to more sensitive case finding. Methods: We studied viral load courses by RT-PCR in oro- and nasopharyngeal swabs, sputum, stool, blood, and urine in nine hospitalized cases. Infectious virus was detected by cell culture. Active replication was demonstrated by analysis of viral subgenomic replicative intermediates. Serology including neutralization testing was done to characterize immune response. Results: Seven cases had upper respiratory tract disease. Lower respiratory tract symptoms seen in two cases were limited. Clinical sensitivity of RT-PCR on swabs taken on days 1-5 of symptoms was 100%, with no differences comparing swab and sputum samples taken simultaneously. Average viral load was 6.76x10E5 copies per swab during the first 5 days. Live virus isolates were obtained from swabs during the first week of illness. Proof of active viral replication in upper respiratory tract tissues was obtained by detection of subgenomic viral RNA. Shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after about one week. Conclusions: The present study shows that COVID-19 can often present as a common cold-like illness. SARS-CoV-2 can actively replicate in the upper respiratory tract, and is shed for a prolonged time after symptoms end, including in stool. These findings suggest adjustments of current case definitions and re-evaluation of the prospects of outbreak containment.

Die feline infektiöse Peritonitis (FIP) zählt zu den bedeutendsten infektiösen Todesursachen bei Katzen und wird durch den letalen Biotyp (FIPV) von felinen Coronaviren (FCoVs) verursacht. Diese FIPVs entstehen aus dem klinisch inapparenten enteralen Biotyp (FECV) durch bisher unbekannte Veränderungen im Genom. Die Erforschung der Pathogenese der FIP wird dadurch erschwert, dass FCoV-Feldviren nicht in vitro kultiviert werden können. Die zur Verfügung stehenden zellkulturadaptierten FCoV-Laborstämme sind zur Aufklärung der molekularen Pathogenese der FIP nicht geeignet. In der vorliegenden Arbeit wurde ein revers-genetisches System für ein FECV etabliert, welches erstmals die Erzeugung rekombinanter FECV-Feldviren ausgehend von einem cDNA-Klon ermöglichte. Folgende Ergebnisse wurden in dieser Arbeit erzielt: 1) Als Grundlage für die Etablierung eines revers-genetischen Systems wurde die gesamte Sequenz eines FECV-Feldvirus bestimmt. Der in dieser Arbeit verwendete Stamm wies den für feline Coronaviren typischen Genomaufbau auf. 2) Um zu zeigen, dass die bestimmte FECV-Sequenz die Replikation in infizierten Zellen ermöglicht, wurde recFECV-S79 hergestellt. Hierzu wurde das Genom des FECV-Feldvirus als cDNA in das Genom des Vacciniavirus, welches als Vektor fungiert, integriert, wobei die Sequenz des S-Gens von einem zellkulturadaptierten FCoV stammte. Ausgehend von diesem infektiösen Klon (vrecFECV-S79) konnten erfolgreich rekombinante FECVs mit heterologem Spikeprotein (recFECV-S79) hergestellt werden. 3) Anschließend wurde ein infektiöser Klon mit dem authentischen S-Gen (vrecFECV) generiert. Die erfolgreiche Herstellung von recFECV wurde mit verschiedenen Verfahren gezeigt. Zunächst wurden Coronavirus-typische Partikel im Elektronenmikroskop detektiert. Mithilfe eines M-Protein-spezifischen monoklonalen Antikörpers konnten diese Partikel in einem Western Blot als FCoVs identifiziert werden. Ein Capsid Protection Assay mit anschließender RT-PCR bzw. qRT-PCR ermöglichte den Nachweis genomischer FCoV-RNA in den gereinigten recFECV-Partikeln. 4) Weiterhin wurde durch Infektion von SPF-Katzen untersucht, ob recFECV in der Lage war, im natürlichen Wirt eine produktive Infektion zu etablieren. Es konnte gezeigt werden, dass infizierte Katzen recFECV über mehr als acht Wochen ausschieden und serokonvertierten. Nach Beendigung des Experiments wurde die Verbreitung von recFECV in unterschiedlichen Organen durch RT-PCR und Immunhistochemie untersucht. Aus den im Tierversuch gewonnenen Daten geht hervor, dass recFECV im natürlichen Wirt eine symptomlose persistente Infektion ähnlich wie bei einer natürlichen FECV-Infektion hervorgerufen hat. Somit stellt das in dieser Arbeit etablierte revers-genetische System für ein FECV-Feldisolat ein ausgezeichnetes Werkzeug zur gezielten Erforschung der molekularen Pathogenese der FIP dar., Feline infectious peritonitis (FIP) belongs tot he most important fatal infectious diseases of cats and it is caused by FIPV which represents the lethal biotype of feline coronavirus (FCoV). FIPVs evolve from the benign enteric biotype (FECV) by so far unidentified mutations. Studies to elucidate the molecular pathogenesis of FIP are limited by the inability to propagate FCoV field viruses in vitro. Though cell culture-adapted laboratory FCoV strains are available, they are unsuitable to study the molecular pathogenesis of FIP. In this study, a reverse genetics system for an FECV was established, which enabled fort he first time the production of recombinant FECV field virus from a cDNA clone. The following results were obtained: 1) Fort he establishment of a reverse genetics system the complete sequence of an FECV field strain was determined. The field virus used in this study followed the typical genome organization of feline coronavirus. 2) To confirm that the sequence determined fort he FECV field strain enabled replication in infected cells, recFECV-S79 was produced. For this purpose, the genome of the FECV field virus containing the S-gene sequence from a cell culture-adapted FCoV was introduced as a cDNA into the vaccinia virus genome which served as a vector. From this infectious clone (vrecFECV-S79), recombinant FECVs with heterologous spike proteins (recFECV-S79) could be successfully recovered.3) Subsequently, an infectious clone with the authentic field isolate S-gene (vrecFECV) was generated. Successful recovery of recFECV was demonstrated by a set of different assays. Particles with the characteristic morphology of coronaviruses could be detected in transmission electron microscopy. Furthermore, Western blot analysis using an anti FCoV-M monoclonal antibody verified the particles as recFECV. Capsid protection assay follwed by RT-PCR or qRT-PCR demonstrated the presence of FECV genomic RNA in purified recFECV particles.4) Furthermore, the ability of recFECV to establish a productive infection in the natural host was tested by infection of SPF cats. It was shown that infected cats seroconverted and shed recFECV continuously for over eight weeks. After termination of the experiment, various organ samples were analyzed fort he presence of FECV by RT-PCR and immunohistochemistry. The animal experiment revealed that recFECV established a symptomless persistent infection in the natural host similar to infections caused by FECV in the field.Taken together, the data presented demonstrated that the reverse genetics system for an FECV field virus established in this study represents an excellent tool to study the molecular pathogenesis of FIP.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

Widespread vaccination programmes led to the global eradication of smallpox, which was certified by the World Health Organisation (WHO), and, since 1978, there has been no case of smallpox anywhere in the world. However, the viable variola virus (VARV), the causative agent of smallpox, is still kept in two maximum security laboratories in Russia and the USA. Despite the eradication of the disease smallpox, clandestine stocks of VARV may exist. In a rapidly changing world, the impact of an intentional VARV release in the human population would nowadays result in a public health emergency of global concern: vaccination programmes were abolished, the percentage of immunosuppressed individuals in the human population is higher, and an increased intercontinental air travel allows for the rapid viral spread of diseases around the world. The WHO has authorised the temporary retention of VARV to enable essential research for public health benefit to take place. This work aims to develop diagnostic tests, antiviral drugs, and safer vaccines. Advances in synthetic biology have made it possible to produce infectious poxvirus particles from chemicals in vitro so that it is now possible to reconstruct VARV. The status of smallpox in the post-eradication era is reviewed.