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Middle East respiratory syndrome coronavirus (MERS-CoV) circulates in dromedary camels in the Arabian Peninsula and occasionally causes spillover infections in humans. MERS-CoV diversity is poorly understood because of the lack of sampling during the COVID-19 pandemic. We collected 558 swab samples from dromedary camels in Saudi Arabia during November 2023–January 2024. We found 39% were positive for MERS-CoV RNA by reverse transcription PCR. We sequenced 42 MERS-CoVs and 7 human 229E-related coronaviruses from camel swab samples by using high-throughput sequencing. Sequences from both viruses formed monophyletic clades apical to recently available genomes. MERS-CoV sequences were most similar to B5 lineage sequences and harbored unique genetic features, including novel amino acid polymorphisms in the spike protein. Further characterization will be required to understand their effects. MERS-CoV spillover into humans poses considerable public health concerns. Our findings indicate surveillance and phenotypic studies are needed to identify and monitor MERS-CoV pandemic potential.

ABSTRACT Wesselsbron virus (WSLV) is a neglected mosquito-borne virus within the yellow fever subgroup in the genus Orthoflavivirus of the Flaviviridae family. Despite being primarily a veterinary pathogen able to cause stillbirths, congenital malformations, and mortality in ruminants, WSLV also infects humans, causing a usually self-limiting febrile illness, or may lead to neurological complications in rare cases. WSLV causes sporadic outbreaks in Southern Africa, but findings in mosquitoes from other African countries suggest a wider distribution. Here, we report the detection and isolation of WSLV from an Aedes mcintoshi mosquito collected in a pristine ecosystem within Semuliki National Park, western Uganda. The detected strain M5937-UG-2018 was impaired in infectivity, replication, and production of infectious particles in cell lines derived from different hosts compared to an epidemic reference strain, SA H177. Full-genome sequencing by next-generation sequencing from the mosquito homogenate revealed a maximum nucleotide identity of 98.1% to a WSLV isolate from a human sample collected in South Africa in 1996. M5937-UG-2018 grouped in phylogenetic analyses with strains from South Africa and Senegal. Reconstruction of the temporal and spatial dispersal of WSLV across Africa estimated a likely origin of WSLV in South Africa in the early 19th century and spread in Southern Africa in the following decades. Long-distance movement toward Western and Eastern Africa was modeled to have occurred in the early 21st century. However, displacing the origin of M5937-UG-2018 did not decrease the likelihood of the model supporting the hypothesis that WSLV is widely distributed in Africa.IMPORTANCEWSLV is a neglected mosquito-borne virus causing teratogenicity in ruminants and febrile illness in humans. WSLV is mainly endemic to Southern Africa, but findings in other regions suggest a wider distribution on the continent. Knowledge of the distribution of WSLV is impaired as differential diagnostics are rarely performed in livestock and humans presenting with symptoms compatible with WSLV infection. Our work investigating viral infections in mosquitoes from a remote tropical rainforest region demonstrates that WSLV is endemic in Uganda. The isolated virus was less infective and showed lower replication ability in vitro compared to an epidemic isolate from South Africa. Phylogeographic reconstruction of spatial and temporal movements, along with the displacement of the origin of the newly detected strain, suggests that WSLV may be widely distributed across Africa. Our data show that the geographic distribution of WSLV and its impact on human and animal health are likely underestimated.

The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin while trypsin protected entry driven by a third S protein from neutralization by S2H97. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and availability of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, can be supported by TMPRSS2-related proteases and may be associated with antibody evasion.

Abstract Anthropogenic disturbances and the subsequent loss of biodiversity are altering species abundances and communities. Since species vary in their pathogen competence, spatio-temporal changes in host assemblages may lead to changes in disease dynamics. We explore how longitudinal changes in bat species assemblages affect the disease dynamics of coronaviruses (CoVs) in more than 2300 cave-dwelling bats captured over two years from five caves in Ghana. This reveals uneven CoV infection patterns between closely related species, with the alpha-CoV 229E-like and SARS-related beta-CoV 2b emerging as multi-host pathogens. Prevalence and infection likelihood for both phylogenetically distinct CoVs is influenced by the abundance of competent species and naïve subadults. Broadly, bat species vary in CoV competence, and highly competent species are more common in less diverse communities, leading to increased CoV prevalence in less diverse bat assemblages. In line with the One Health framework, our work supports the notion that biodiversity conservation may be the most proactive measure to prevent the spread of pathogens with zoonotic potential.

Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in dromedaries in Africa, but camel-to-human transmission is limited. Sustained 12-month sampling of dromedaries in a Kenya abattoir hub showed biphasic MERS-CoV incidence; peak detections occurred in October 2022 and February 2023. Dromedary-exposed abattoir workers (7/48) had serologic signs of previous MERS-CoV exposure.

Setting up a global SARS-CoV-2 surveillance system requires an understanding of how virus isolation and propagation practices, use of animal or human sera, and different neutralisation assay platforms influence assessment of SARS-CoV-2 antigenicity. In this study, with the contribution of 15 independent laboratories across all WHO regions, we carried out a controlled analysis of neutralisation assay platforms using the first WHO International Standard for antibodies to SARS-CoV-2 variants of concern (source: NIBSC). Live virus isolates (source: WHO BioHub or individual labs) or spike plasmids (individual labs) for pseudovirus production were used to perform neutralisation assays using the same serum panels. When comparing fold drops, excellent data consistency was observed across the labs using common reagents, including between pseudovirus and live virus neutralisation assays (RMSD of data from mean fold drop was 0.59). Utilising a Bayesian model, geometric mean titres and assay titre magnitudes (offsets) can describe the data efficiently. Titre magnitudes were seen to vary largely even for labs within the same assay group. We have observed that overall, live Microneutralisation assays tend to have the lowest titres, whereas Pseudovirus Neutralisation have the highest (with a mean difference of 3.2 log2 units between the two). These findings are relevant for laboratory networks, such as the WHO Coronavirus Laboratory Network (CoViNet), that seek to support a global surveillance system for evolution and antigenic characterisation of variants to support monitoring of population immunity and vaccine composition policy.

IntroductionOxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear.MethodsIn this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins.ResultsOur findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation—a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1–1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1–1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization.ConclusionOur study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.

Abstract Anthropogenic disturbance may increase the emergence of zoonoses. Especially generalists that cope with disturbance and live in close contact with humans and livestock may become reservoirs of zoonotic pathogens. Yet, whether anthropogenic disturbance modifies host-pathogen co-evolutionary relationships in generalists is unknown. We assessed pathogen diversity, neutral genome-wide diversity (SNPs) and adaptive MHC class II diversity in a rodent generalist inhabiting three lowland rainforest landscapes with varying anthropogenic disturbance, and determined which MHC alleles co-occurred more frequently with 13 gastrointestinal nematodes, blood trypanosomes, and four viruses. Pathogen-specific selection pressures varied between landscapes. Genome-wide diversity declined with the degree of disturbance, while MHC diversity was only reduced in the most disturbed landscape. Furthermore, pristine forest landscapes had more functional important MHC–pathogen associations when compared to disturbed forests. We show co-evolutionary links between host and pathogens impoverished in human-disturbed landscapes. This underscores that parasite-mediated selection might change even in generalist species following human disturbance which in turn may facilitate host switching and the emergence of zoonoses.

Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.

Abstract Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Abstract Background Intrinsic fitness costs are likely to have guided the selection of lineage-determining mutations during emergence of variants of SARS-CoV-2. Whereas changes in receptor affinity and antibody neutralization have been thoroughly mapped for individual mutations in spike, their influence on intrinsic replicative fitness remains understudied. Methods We analyzed mutations in immunodominant spike epitope E484 that became temporarily fixed over the pandemic. We engineered the resulting immune escape mutations E484K, -A, and -Q in recombinant SARS-CoV-2. We characterized viral replication, entry, and competitive fitness with and without immune serum from humans with defined exposure/vaccination history and hamsters monospecifically infected with the E484K variant. We additionally engineered a virus containing the Omicron signature mutations N501Y and Q498R that were predicted to epistatically enhance receptor binding. Results Multistep growth kinetics in Vero-, Calu-3, and NCI-H1299 were identical between viruses. Synchronized entry experiments based on cold absorption and temperature shift identified only an insignificant trend toward faster entry of the E484K variant. Competitive passage experiments revealed clear replicative fitness differences. In absence of immune serum, E484A and E484Q, but not E484K, were replaced by wildtype (WT) in competition assays. In presence of immune serum, all three mutants outcompeted WT. Decreased E484A fitness levels were over-compensated for by N501Y and Q498R, identifying a putative Omicron founder background that exceeds the intrinsic and effective fitness of WT and matches that of E484K. Critically, the E484A/Q498R/N501Y mutant and E484K have equal fitness also in presence of pre-Omicron vaccinee serum, whereas the fitness gain by E484K is lost in the presence of serum raised against the E484K variant in hamsters. Conclusions The emergence of E484A and E484Q prior to widespread population immunity may have been limited by fitness costs. In populations already exposed to the early immune escape epitope E484K, the Omicron founder background may have provided a basis for alternative immune escape evolution via E484A. Studies of major antigenic epitope changes with and without their epistatic context help reconstruct the sequential adjustments of intrinsic fitness versus neutralization escape during the evolution of major SARS-CoV-2 variants in an increasingly immune human population.

Abstract The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 106 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.

Abstract The SARS-CoV-2 Omicron subvariants BA.1 and BA.2 exhibit reduced lung cell infection relative to previously circulating SARS-CoV-2 variants, which may account for their reduced pathogenicity. However, it is unclear whether lung cell infection by BA.5, which displaced these variants, remains attenuated. Here, we show that the spike (S) protein of BA.5 exhibits increased cleavage at the S1/S2 site and drives cell-cell fusion and lung cell entry with higher efficiency than its counterparts from BA.1 and BA.2. Increased lung cell entry depends on mutation H69Δ/V70Δ and is associated with efficient replication of BA.5 in cultured lung cells. Further, BA.5 replicates in the lungs of female Balb/c mice and the nasal cavity of female ferrets with much higher efficiency than BA.1. These results suggest that BA.5 has acquired the ability to efficiently infect lung cells, a prerequisite for causing severe disease, suggesting that evolution of Omicron subvariants can result in partial loss of attenuation.

Bombali virus (BOMV) is a novel Orthoebolavirus that has been detected in free-tailed bats in Sierra Leone, Guinea, Kenya, and Mozambique. We screened our collection of 349 free-tailed bat lungs collected in Côte d’Ivoire and Tanzania for BOMV RNA and tested 228 bat blood samples for BOMV antibodies. We did not detect BOMV-specific antibodies but found BOMV RNA in a Mops condylurus bat from Tanzania, marking the first detection of an ebolavirus in this country. Our findings further expand the geographic range of BOMV and support M. condylurus’ role as a natural BOMV host.

Objective: To explore a possible connection between active viral infections and manifestation of Dermatomyositis (DM). Methods: Skeletal muscle biopsies were analyzed from patients diagnosed with juvenile (n=10) and adult (n=12) DM. Adult DM patients harbored autoantibodies against either TIF-1γ (n=7) or MDA5 (n=5). Additionally, we investigated skeletal muscle biopsies from non-diseased controls (NDC, n=5). We used an unbiased high-throughput sequencing (HTS) approach to detect viral sequences. To further increase sequencing depth, a host depletion approach was applied. Results: In this observational study, no relevant viral sequences were detected either by native sequencing or after host depletion. The absence of detectable viral sequences makes an active viral infection of the muscle tissue unlikely to be the cause of DM in our cohorts. Discussion: Type I interferons (IFN) play a major role in the pathogenesis of both juvenile and adult dermatomyositis (DM). The IFN response is remarkably conserved between DM subtypes classified by specific autoantibodies. Certain acute viral infections are accompanied by a prominent type I IFN response involving similar downstream mechanisms as in DM. Aiming to elucidate the pathogenesis of DM in skeletal muscle tissue, we used an untargeted high-throughput sequencing and a host depletion approach to detect possible causative viruses.

Pneumonia is the leading cause of death in children, however, the microbial aetiology of pneumonia is not well elucidated in low- and middle-income countries. Our study was aimed at determining the microbial aetiologies of childhood pneumonia and associated risk factors in HIV and non-HIV infected children. We conducted a case-control study that enrolled children with pneumonia as cases and non-pneumonia as controls from July 2017 to May 2020. Induced sputum and blood samples were investigated for microbial organisms using standard microbiological techniques. DNA/RNA was extracted from sputum samples and tested for viral and bacterial agents. Four hundred and four (404) subjects consisting of 231 (57.2%) cases and 173 (42.8%) controls were enrolled. We identified a significant (p = 0.011) proportion of viruses in cases (125; 54.1%, 95%CI: 47.4-60.7) than controls (71; 33.6%, 95%CI: 33.6-48.8) and these were mostly contributed to by Respiratory Syncytial Virus. Staphylococcus aureus (16; 4.0%), Klebsiella spp. (15, 3.7%) and Streptococcus pneumoniae (8, 2.0%) were the main bacterial agents identified in sputum or induced sputum samples. HIV infected children with viral-bacterial co-detection were found to have very severe pneumonia compared to those with only viral or bacterial infection. Indoor cooking (OR = 2.36; 95%CI:1.41-3.96) was found to be associated with pneumonia risk in patients. This study demonstrates the importance of various microbial pathogens, particularly RSV, in contributing to pneumonia in HIV and non-HIV paediatric populations. There is a need to accelerate clinical trials of RSV vaccines in African populations to support improvement of patient care.

Since late 2020, SARS-CoV-2 variants have regularly emerged with competitive and phenotypic differences from previously circulating strains, sometimes with the potential to escape from immunity produced by prior exposure and infection. The Early Detection group is one of the constituent groups of the US National Institutes of Health National Institute of Allergy and Infectious Diseases SARS-CoV-2 Assessment of Viral Evolution program. The group uses bioinformatic methods to monitor the emergence, spread, and potential phenotypic properties of emerging and circulating strains to identify the most relevant variants for experimental groups within the program to phenotypically characterize. Since April 2021, the group has prioritized variants monthly. Prioritization successes include rapidly identifying most major variants of SARS-CoV-2 and providing experimental groups within the National Institutes of Health program easy access to regularly updated information on the recent evolution and epidemiology of SARS-CoV-2 that can be used to guide phenotypic investigations.

Abstract Respiratory infections are one of the most common causes of death among children under the age of five years. Data on prevalence and relevance of specific organisms in African children are still lacking. This case–control-study investigated prevalence and relevance of specific organisms in Ghanaian children admitted to hospital with symptoms of lower respiratory tract infection (LRTI). Pharyngeal swabs were taken and tested by PCR for 19 respiratory isolates. Adjusted odds ratios (aORs) were calculated to estimate associations between isolates and admission with LRTI. Population attributable fractions (PAFs) were calculated to assess the proportion of LRTI cases due to a particular pathogen. The study included 327 cases and 562 controls. We found associations between detection and admission for LRTI for influenza (aOR 98.6; 95% confidence interval (CI) 20.0–1789.6), respiratory syncytial virus (aOR 40.2; 95% CI 7.2–758.6), H. influenzae (aOR 4.1; 95% CI 2.2–7.9) and S. pneumoniae (aOR 2.4; 95% CI 1.7–3.4). PAFs ≥ 10% were observed for S. pneumoniae (30%; 95% CI 26–42), H. influenzae (10%; 95% CI 2–19) and influenza (10%; 95% CI 2–18). This study highlights the need for heightened surveillance and development of effective vaccines for respiratory pathogens other than SARS-CoV-2 in the future.

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19.

SARS-CoV-2 Mu variant emerged in Colombia in 2021 and spread globally. In 49 serum samples from vaccinees and COVID-19 survivors in Colombia, neutralization was significantly lower (p

Abstract Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.

In a clinical trial, Fathi et al. show that a booster vaccination with a vector vaccine candidate against the highly pathogenic Middle East Respiratory Syndrome coronavirus is safe and strongly improves the immunity generated by primary immunization.

Abstract Background Encephalitis is a serious disease of the brain characterized by prodromal and specific neurological symptoms. HIV infections offer opportunistic viruses, such as Varicella-zoster virus (VZV), the chance to cause encephalitis in patients. There is a lack of information on the genetic diversity of VZV in Ghana and other parts of Africa which requires sequencing and characterization studies to address. The active evolution of HIV-1 in West Africa also requires continuous surveillance for the emergence of new genetic forms. Case presentation VZV was detected in the CSF sample of an 11-year-old patient presenting with symptoms of encephalitis by real-time PCR diagnostics. To identify possible unknown aetiological pathogens, next-generation sequencing was performed, and revealed an HIV-1 co-infection. Alignments of concatenated HIV-1 genome fragments in the gag, pol, vif, env and nef regions and a near-complete VZV genome were analyzed by Bayesian inference, and phylogenetic trees were generated. The VZV sequence belongs to clade 5 and the HIV-1 sequence is a member of the CRF02_AG predominant circulating recombinant form in Ghana. Conclusions Diagnostic tests for CSF HIV would be useful where possible in patients presenting with encephalitis due to VZV and other opportunistic viruses in Kumasi to shed light on the role of HIV in encephalitis cases in Ghana. This report reaffirms the role of the CRF02_AG circulating recombinant form in HIV infections in Ghana and also gives a preliminary genetic characterization of VZV in Kumasi, Ghana.

Children are less likely to be infected with SARS-CoV-2 and develop less severe disease than adults, which makes estimation of infection rates challenging. Here, the authors conduct seroprevalence surveys of children in Germany, describe changes in prevalence over time, and identify risk factors for infection.

Genomic analyses of spike glycoprotein genes of European bat SARS-related coronaviruses suggest that furin cleavage sites can be acquired in the bat reservoir via conserved molecular mechanisms, supporting a natural origin of SARS-CoV-2.

Abstract Camels gained attention since the discovery of MERS-CoV as intermediary hosts for potentially epidemic zoonotic viruses. DcHEV is a novel zoonotic pathogen associated with camel contact. This study aimed to genetically characterize DcHEV in domestic and imported camels in Saudi Arabia. DcHEV was detected by RT-PCR in serum samples, PCR-positive samples were subjected to sequencing and phylogenetic analyses. DcHEV was detected in 1.77% of samples with higher positivity in domestic DCs. All positive imported dromedaries were from Sudan with age declining prevalence. Domestic DcHEV sequences clustered with sequences from Kenya, Somalia, and UAE while imported sequences clustered with one DcHEV isolate from UAE and both sequences clustered away from isolates reported from Pakistan. Full-genome sequences showed 24 amino acid difference with reference sequences. Our results confirm the detection of DcHEV in domestic and imported DCs. Further investigations are needed in human and camel populations to identify DcHEV potential zoonosis threat.

Summary: Opposing effects of interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) on SARS-CoV-2 infection have been reported. The reasons for this are unclear and the role of IFITMs in infection of other human coronaviruses (hCoVs) remains poorly understood. Here, we demonstrate that endogenous expression of IFITM2 and/or IFITM3 is critical for efficient replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 but has little effect on MERS-, NL63-and 229E-hCoVs. In contrast, overexpression of IFITMs inhibits all these hCoVs, and the corresponding spike-containing pseudo-particles, except OC43, which is enhanced by IFITM3. We further demonstrate that overexpression of IFITMs impairs cell surface expression of ACE2 representing the entry receptor of SARS-CoVs and hCoV-NL63 but not hCoV-OC43. Our results explain the inhibitory effects of artificial IFITM overexpression on ACE2-tropic SARS-CoVs and show that three hCoVs, including major causative agents of severe respiratory disease, hijack IFITMs for efficient infection of human cells.

Severe acute respiratory syndrome coronavirus 2 Delta variant epidemiology in Africa is unknown. We found Delta variant was introduced in Benin during April–May 2021 and became predominant within 2 months, after which a steep increase in reported coronavirus disease incidence occurred. Benin might require increased nonpharmaceutical interventions and vaccination coverage.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., “arboviruses”), infecting a certain type of host (e.g., “mycoviruses,” “bacteriophages”) or displaying specific pathogenicity (e.g., “human immunodeficiency viruses”), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent. Transforming an existing phenotypic classification of viruses into one based on evolutionary relationships that can accommodate the vast number of viruses characterized in metagenomics and environmental studies is an ongoing challenge. This Consensus View explains how such a taxonomy can be expanded to encapsulate viral diversity and to recognize independent biological origins of different virus groups.

Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa.

MERS-CoV is enzootic in dromedary camels, can spread to humans but undergoes limited onward transmission. Here, Schroeder et al. compare clinical isolates of MERS-CoV in vitro and show that the predominantly circulating recombinant lineage 5 possess a fitness advantage over parental lineage 3 and 4 due to reduced activation of innate immune signaling.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

We detected delayed and reduced antibody and T-cell responses after BNT162b2 vaccination in 71 elderly persons (median age 81 years) compared with 123 healthcare workers (median age 34 years) in Germany. These data emphasize that nonpharmaceutical interventions for coronavirus disease remain crucial and that additional immunizations for the elderly might become necessary.

The immune response is key in determining disease severity of COVID19. Here Nouailles et al., apply bulk proteomics and scRNA-Seq of lung and blood samples of SARS-CoV-2 infected Syrian hamsters and provide a temporal atlas of the systemic and pulmonary cellular responses.

One week after second vaccinations were administered, an outbreak of B.1.1.7 lineage severe acute respiratory syndrome coronavirus 2 infections occurred in a long-term care facility in Berlin, Germany, affecting 16/20 vaccinated and 4/4 unvaccinated residents. Despite considerable viral loads, vaccinated residents experienced mild symptoms and faster time to negative test results.

Abstract Background Hepatitis E virus (HEV) is among the leading causes of viral hepatitis in most developing countries. Zoonotic acquisition of HEV genotype 3 from swine has come into focus more recently. Available studies on HEV in Ghana and other countries in the region do not provide enough information towards understanding the epidemiology of HEV in human and animal populations. Towards this end, we conducted a comparative cross-sectional study to determine the seroprevalence and risk factors associated with HEV exposure, both in swine and humans working on pig farms in typical local settings. The presence of viral RNA in human and swine samples was also evaluated, along with classification of viral sequences from HEV-positive samples. Methods Structured questionnaires soliciting information on pigs reared, as well as socio-demographic information including age, sex and educational background of humans was collected. A total of 10 ml and 5 ml of whole blood was collected from pigs and human participants respectively. ELISA and real-time RT-PCR were performed on the sera for the qualitative detection of IgG antibodies to hepatitis E virus and viral RNA, respectively. Results Five hundred and forty-four (544) human participants including 264 swine contacts and 280 swine non-contacts were enrolled in the study. Although the proportion of HEV IgG antibodies was higher in contact groups (114; 54.3%) than non-contact groups (96; 45.7%), a multivariate analysis did not show any significant difference. No HEV RNA was detected in human samples. Similarly, 720 pigs were sampled from 18 farms located in five regions in Ghana. Twenty-three (23) of the pigs (3.2, 95%CI = 2.0–4.8) were positive for HEV RNA by real-time RT-PCR testing. Sequences obtained from HEV-positive samples were found to share high sequence identities with each other and clustered with other genotype 3 viruses indicating the existence of circulating zoonotic genotype 3 viruses on farms. Although we did not find evidence of pig to human transmission of HEV genotype 3, the presence of this genotype in pigs shows the potential for possible zoonotic transmission in African farm settings and buttresses the importance of active surveillance for the infection among at risk populations.

Viruses manipulate host cell pathways to support infection. Here the authors show that SARS-CoV-2 infection modulates cellular metabolism and limits autophagy, and identify druggable host pathways for virus inhibition.

Population-based studies of SARS-CoV-2 seroprevalence are needed to understand levels of immunity and antibody dynamics. Here, the authors show that the seroprevalence in Bonn, Germany was low (

ABSTRACT SARS-CoV-2 variants of concern (VOC) acquired mutations in the spike (S) protein, including E484K, that confer resistance to neutralizing antibodies. However, it is incompletely understood how these mutations impact viral entry into host cells. Here, we analyzed how mutations at position 484 that have been detected in COVID-19 patients impact cell entry and antibody-mediated neutralization. We report that mutation E484D markedly increased SARS-CoV-2 S-driven entry into the hepatoma cell line Huh-7 and the lung cell NCI-H1299 without augmenting ACE2 binding. Notably, mutation E484D largely rescued Huh-7 but not Vero cell entry from blockade by the neutralizing antibody Imdevimab and rendered Huh-7 cell entry ACE2-independent. These results suggest that the naturally occurring mutation E484D allows SARS-CoV-2 to employ an ACE2-independent mechanism for entry that is largely insensitive against Imdevimab, an antibody employed for COVID-19 therapy. IMPORTANCE The interaction of the SARS-CoV-2 spike protein (S) with the cellular receptor ACE2 is considered essential for infection and constitutes the key target for antibodies induced upon infection and vaccination. Here, using a surrogate system for viral entry, we provide evidence that a naturally occurring mutation can liberate SARS-CoV-2 from ACE2-dependence and that ACE2-independent entry may protect the virus from neutralization by an antibody used for COVID-19 therapy.

We report an outbreak of coronavirus disease with 74 cases related to a nightclub in Germany in March 2020. Staff members were particularly affected (attack rate 56%) and likely caused sustained viral transmission after an event at the club. This outbreak illustrates the potential for superspreader events and corroborates current club closures.

At a regional and local level, the COVID-19 pandemic has not spread out uniformly and some German municipalities have been particularly affected. The seroepidemiological data from these areas helps estimate the proportion of the population that has been infected with SARS-CoV-2 (seroprevalence), as well as the number of undetected infections and asymptomatic cases. In four municipalities which were especially affected, 2,000 participants will be tested for an active SARS-CoV-2 infection (oropharyngeal swab) or a past infection (blood specimen IgG antibody test). Participants will also be asked to fill out a short written questionnaire at study centres and complete a follow-up questionnaire either online or by telephone, including information on issues such as possible exposure, susceptability, symptoms and medical history. The CORONA-MONITORING lokal study will allow to determine the proportion of the population with SARS-CoV-2 antibodies in four particularly affected locations. This study will increase the accuracy of estimates regarding the scope of the epidemic, help determine risk and protective factors for an infection and therefore also identify especially exposed groups and, as such, it will be crucial towards planning of prevention measures.

Abstract Background Apart from the huge worldwide economic losses often occasioned by bovine coronavirus (BCoV) to the livestock industry, particularly with respect to cattle rearing, continuous surveillance of the virus in cattle and small ruminants is essential in monitoring variations in the virus that could enhance host switching. In this study, we collected rectal swabs from a total of 1,498 cattle, sheep and goats. BCoV detection was based on reverse transcriptase polymerase chain reaction. Sanger sequencing of the partial RNA-dependent RNA polymerase (RdRp) region for postive samples were done and nucleotide sequences were compared with homologous sequences from the GenBank. Results The study reports a BCoV prevalence of 0.3%, consisting of 4 positive cases; 3 goats and 1 cattle. Less than 10% of all the animals sampled showed clinical signs such as diarrhea and respiratory distress except for high temperature which occurred in > 1000 of the animals. However, none of the 4 BCoV positive animals manifested any clinical signs of the infection at the time of sample collection. Bayesian majority-rule cladogram comparing partial and full length BCoV RdRp genes obtained in the study to data from the GenBank revealed that the sequences obtained from this study formed one large monophyletic group with those from different species and countries. The goat sequences were similar to each other and clustered within the same clade. No major variations were thus observed between our isolates and those from elsewhere. Conclusions Given that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

Orthohepevirus A genotype 7 is a novel zoonotic variant of hepatitis E virus. To clarify infection in the animal reservoir, we virologically monitored 11 dromedary dam–calf pairs. All calves became infected during the first 6 months of life and cleared the virus after an average of 2 months. Dams did not become infected.

Abstract Background Hepatitis E virus (HEV) is a major cause of human hepatitis worldwide. Zoonotic genotypes of the virus have been found in diverse animal species with pigs playing a major role. Putative risk of zoonotic infection from livestock particularly swine in Sub-Saharan Africa including Ghana is poorly understood due to scarcity of available data, especially HEV sequence information. Methods Serum samples were collected from cattle, sheep, goats and pigs from Kumasi in the Ashanti region of Ghana. Samples were subjected to nested RT-PCR screening and quantification of HEV RNA-positive samples using real-time RT-PCR and the World Health Organization International Standard for HEV. Testing of all pig samples for antibodies was done by ELISA. Sanger sequencing and genotyping was performed and one representative complete genome was generated to facilitate genome-wide comparison to other available African HEV sequences by phylogenetic analysis. Results A total of 420 samples were available from cattle (n = 105), goats (n = 124), pigs (n = 89) and sheep (n = 102). HEV Viral RNA was detected only in pig samples (10.1%). The antibody detection rate in pigs was 77.5%, with positive samples from all sampling sites. Average viral load was 1 × 105 (range 1.02 × 103 to 3.17 × 105) International Units per mL of serum with no statistically significant differences between age groups (≤ 6 month, > 6 months) by a T-test comparison of means (t = 1.4272, df = 7, p = 0.1966). Sequences obtained in this study form a monophyletic group within HEV genotype 3. Sequences from Cameroon, Ghana, Burkina Faso and Madagascar were found to share a most recent common ancestor; however this was not the case for other African HEV sequences. Conclusion HEV genotype 3 is highly endemic in pigs in Ghana and likely poses a zoonotic risk to people exposed to pigs. HEV genotype 3 in Ghana shares a common origin with other virus strains from Sub-Saharan Africa.

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein–specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2–infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2–specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Infection control instructions call for use of alcohol-based hand rub solutions to inactivate severe acute respiratory syndrome coronavirus 2. We determined the virucidal activity of World Health Organization–recommended hand rub formulations, at full strength and multiple dilutions, and of the active ingredients. All disinfectants demonstrated efficient virus inactivation.

Carnivores such as cats and minks are highly susceptible to SARS-CoV-2. Brazil is a global COVID-19 hot spot and several cases of human-to-cat transmission have been documented. We investigated the spread of SARS-CoV-2 by testing 547 domestic cats sampled between July-November 2020 from seven states in southern, southeastern, and northeastern Brazil. Moreover, we investigated whether immune responses elicited by enzootic coronaviruses affect SARS-CoV-2 infection in cats. We found infection with significantly higher neutralizing antibody titers against the Gamma variant of concern, endemic in Brazil during 2020, than against an early SARS-CoV-2 B.1 isolate (p