Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Allicin (diallyl thiosulfinate) is the major thiol-reactive organosulfur compound produced by garlic plants (Allium sativum) upon tissue damage. Allicin exerts its strong antimicrobial activity against bacteria and fungi via S-thioallylation of protein thiols and low molecular weight thiols. Here, we investigated the effect of allicin on SARS-CoV-2 infected Vero E6 and Calu-3 cells. Toxicity tests revealed that Calu-3 cells showed greater allicin tolerance, probably due to >4-fold higher GSH levels compared to the very sensitive Vero E6 cells. Exposure of infected Vero E6 and Calu-3 cells to biocompatible allicin doses led to a ∼60-70% decrease of viral RNA and infectious viral particles. Label-free quantitative proteomics was used to investigate the changes in the Calu-3 proteome after SARS-CoV-2 infection and the effect of allicin on the host-virus proteome. SARS-CoV-2 infection of Calu-3 cells caused a strong induction of the antiviral interferon-stimulated gene (ISG) signature, including several antiviral effectors, such as cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, OAS, and ISG15, pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTCL1, and ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin treatment of infected Calu-3 cells reduced the expression of IFN signaling pathways and ISG effectors and reverted several host pathways to levels of uninfected cells. Allicin further reduced the abundance of the structural viral proteins N, M, S and ORF3 in the host-virus proteome. In conclusion, our data demonstrate the antiviral and immunomodulatory activity of biocompatible doses of allicin in SARS-CoV-2-infected cell cultures. Future drug research should be directed to exploit the thiol-reactivity of allicin derivatives with increased stability and lower human cell toxicity as antiviral lead compounds.

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

The recent COVID-19 pandemic has demonstrated again the global threat posed by emerging zoonotic coronaviruses. During the past two decades alone, humans have experienced the emergence of several coronaviruses, such as SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019. To date, MERS-CoV has been detected in 27 countries, with a case fatality ratio of approximately 34.5%. Similar to other coronaviruses, MERS-CoV presumably originated from bats; however, the main reservoir and primary source of human infections are dromedary camels. Other species within the Camelidae family, such as Bactrian camels, alpacas, and llamas, seem to be susceptible to the infection as well, although to a lesser extent. In contrast, susceptibility studies on sheep, goats, cattle, pigs, chickens, and horses obtained divergent results. In the present study, we tested nasal swabs and/or sera from 55 sheep, 45 goats, and 52 cattle, collected at the largest livestock market in the United Arab Emirates, where dromedaries are also traded, for the presence of MERS-CoV nucleic acid by RT-qPCR, and for specific antibodies by immunofluorescence assay. All sera were negative for MERS-CoV-reactive antibodies, but the nasal swab of one sheep (1.8%) repeatedly tested positive for MERS-CoV nucleic acid. Next generation sequencing (NGS) of the complete N gene of the sheep-derived MERS-CoV revealed >99% nucleotide identity to MERS-CoV sequences of five dromedaries in nearby pens and to three reference sequences. The NGS sequence of the sheep-derived MERS-CoV was confirmed by conventional RT-PCR of a part of the N gene and subsequent Sanger sequencing. All MERS-CoV sequences clustered within clade B, lineage 5. In conclusion, our study shows that noncamelid livestock, such as sheep, goats, and cattle do not play a major role in MERS-CoV epidemiology. The one sheep that tested positive most likely reflects an accidental viral spillover event from infected dromedaries in nearby pens.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)–reactive and SARS-CoV-2–cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830–reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti–SARS-CoV-2-S1-IgG antibodies. Spike–cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike–cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur.

The inability of patients with CVID to mount specific antibody responses to pathogens has raised concerns on the risk and severity of SARS-CoV-2 infection, but there might be a role for protective T cells in these patients. SARS-CoV-2 reactive T cells have been reported for SARS-CoV-2 unexposed healthy individuals. Until now, there is no data on T cell immunity to SARS-CoV-2 infection in CVID. This study aimed to evaluate reactive T cells to human endemic corona viruses (HCoV) and to study pre-existing SARS-CoV-2 reactive T cells in unexposed CVID patients. We evaluated SARS-CoV-2- and HCoV-229E and -OC43 reactive T cells in response to seven peptide pools, including spike and nucleocapsid (NCAP) proteins, in 11 unexposed CVID, 12 unexposed and 11 post COVID-19 healthy controls (HC). We further characterized reactive T cells by IFNγ, TNFα and IL-2 profiles. SARS-CoV-2 spike-reactive CD4+ T cells were detected in 7 of 11 unexposed CVID patients, albeit with fewer multifunctional (IFNγ/TNFα/IL-2) cells than unexposed HC. CVID patients had no SARS-CoV-2 NCAP reactive CD4+ T cells and less reactive CD8+ cells compared to unexposed HC. We observed a correlation between T cell reactivity against spike of SARS-CoV-2 and HCoVs in unexposed, but not post COVID-19 HC, suggesting cross-reactivity. T cell responses in post COVID-19 HC could be distinguished from unexposed HC by higher frequencies of triple-positive NCAP reactive CD4+ T cells. Taken together, SARS-CoV-2 reactive T cells are detectable in unexposed CVID patients albeit with lower recognition frequencies and polyfunctional potential. Frequencies of triple-functional reactive CD4+ cells might provide a marker to distinguish HCoV cross-reactive from SARS-CoV-2 specific T cell responses. Our data provides evidence, that anti-viral T cell immunity is not relevantly impaired in most CVID patients.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels.

Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2.

Avian bornaviruses are a genetically diverse group of viruses initially discovered in 2008. They are known to infect several avian orders. Bornaviruses of parrots and related species (Psittaciformes) are causative agents of proventricular dilatation disease, a chronic and often fatal neurologic disease widely distributed in captive psittacine populations. Although knowledge has considerably increased in the past years, many aspects of the biology of avian bornaviruses are still undiscovered. In particular, the precise way of transmission remains unknown.

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused human respiratory infections with a high case fatality rate since 2012. However, the mode of virus transmission is not well understood. The findings of epidemiological and virological studies prompted us to hypothesize that the human gastrointestinal tract could serve as an alternative route to acquire MERS-CoV infection. We demonstrated that human primary intestinal epithelial cells, small intestine explants, and intestinal organoids were highly susceptible to MERS-CoV and can sustain robust viral replication. We also identified the evidence of enteric MERS-CoV infection in the stool specimen of a clinical patient. MERS-CoV was considerably resistant to fed-state gastrointestinal fluids but less tolerant to highly acidic fasted-state gastric fluid. In polarized Caco-2 cells cultured in Transwell inserts, apical MERS-CoV inoculation was more effective in establishing infection than basolateral inoculation. Notably, direct intragastric inoculation of MERS-CoV caused a lethal infection in human DPP4 transgenic mice. Histological examination revealed MERS-CoV enteric infection in all inoculated mice, as shown by the presence of virus-positive cells, progressive inflammation, and epithelial degeneration in small intestines, which were exaggerated in the mice pretreated with the proton pump inhibitor pantoprazole. With the progression of the enteric infection, inflammation, virus-positive cells, and live viruses emerged in the lung tissues, indicating the development of sequential respiratory infection. Taken together, these data suggest that the human intestinal tract may serve as an alternative infection route for MERS-CoV.

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Thailand reported the first Middle East respiratory syndrome (MERS) case on 18 June 2015 (day 4) in an Omani patient with heart condition who was diagnosed with pneumonia on hospital admission on 15 June 2015 (day 1). Two false negative RT-PCR on upper respiratory tract samples on days 2 and 3 led to a 48-hour diagnosis delay and a decision to transfer the patient out of the negative pressure unit (NPU). Subsequent examination of sputum later on day 3 confirmed MERS coronavirus (MERS-CoV) infection. The patient was immediately moved back into the NPU and then transferred to Bamrasnaradura Infectious Disease Institute. Over 170 contacts were traced; 48 were quarantined and 122 self-monitored for symptoms. High-risk close contacts exhibiting no symptoms, and whose laboratory testing on the 12th day after exposure was negative, were released on the 14th day. The Omani Ministry of Health (MOH) was immediately notified using the International Health Regulation (IHR) mechanism. Outbreak investigation was conducted in Oman, and was both published on the World Health Organization (WHO) intranet and shared with Thailand's IHR focal point. The key to successful infection control, with no secondary transmission, were the collaborative efforts among hospitals, laboratories and MOHs of both countries.

Despite RT-PCR confirmed COVID-19, specific antibodies to SARS-CoV-2 spike are undetectable in serum in approximately 10% of convalescent patients after mild disease course. This raises the question of induction and persistence of SARS-CoV-2-reactive T cells in these convalescent individuals. Using flow cytometry, we assessed specific SARS-CoV-2 and human endemic coronaviruses (HCoV-229E, -OC43) reactive T cells after stimulation with spike and nucleocapsid peptide pools and analyzed cytokine polyfunctionality (IFNγ, TNFα, and IL-2) in seropositive and seronegative convalescent COVID-19 patients as well as in unexposed healthy controls. Stimulation with SARS-CoV-2 spike and nucleocapsid (NCAP) as well as HCoV spike peptide pools elicited a similar T cell response in seropositive and seronegative post COVID-19 patients. Significantly higher frequencies of polyfunctional cytokine nucleocapsid reactive CD4+ T cells (triple positive for IFNγ, TNFα, and IL-2) were observed in both, seropositive (p = 0.008) and seronegative (p = 0.04), COVID-19 convalescent compared to healthy controls and were detectable up to day 162 post RT-PCR positivity in seronegative convalescents. Our data indicate an important role of NCAP-specific T cells for viral control.

IntroductionNumerous CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) are offered in Europe, several of them with unconfirmed quality claims.AimWe performed an independent head-to-head evaluation of the sensitivity of SARS-CoV-2 Ag RDT offered in Germany.MethodsWe addressed the sensitivity of 122 Ag RDT in direct comparison using a common evaluation panel comprised of 50 specimens. Minimum sensitivity of 75% for panel specimens with a PCR quantification cycle (Cq) ≤ 25 was used to identify Ag RDT eligible for reimbursement in the German healthcare system.ResultsThe sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with Cq ≤ 25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which failed completely. Some RDT exhibited high sensitivity, e.g. 97.5 % for Cq < 30.ConclusionsThis comparative evaluation succeeded in distinguishing less sensitive from better performing Ag RDT. Most of the evaluated Ag RDT appeared to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen endemic to the Arabian Peninsula. Dromedary camels are a likely source of infection and the virus probably originated in Africa. We studied the genetic diversity, geographical structure, infection prevalence, and age-associated prevalence among camels at the largest entry port of camels from Africa into the Arabian Peninsula.

International travel is a major driver of the introduction and spread of SARS-CoV-2.