Genome sequencing of the mpox virus 2022 outbreak with amplicon-based Oxford Nanopore MinION sequencing.

We present an amplicon-based assay for MinION Nanopore sequencing of mpox virus (MPXV) genomes from clinical specimens, obtaining high-quality results with an average genome coverage of 99% for Ct values of up to 25, and a genome coverage of 97.1% for Ct values from 25 to 30 which are challenging to sequence. This assay is easy to implement in PCR-based workflows and provides accurate genomic data within a short time.

Serological methods for the detection of antibodies against monkeypox virus applicable for laboratories with different biosafety levels.

The monkeypox virus (MPXV) outbreak in 2022 has renewed interest in the detection of antibodies against orthopox viruses (OPXV) and MPXV, as serological methods can aid diagnostics and are key to epidemiological studies. Here three complementary serological methods are described with different strengths to aid the development and evaluation of in-house assays: An immunofluorescence assay (IFA) for specific detection of IgG and IgM, an enzyme-linked immunosorbent assay for higher sample throughput to aid epidemiological studies and a neutralization test to detect virus neutralizing antibodies. As implementation of MPXV-specific diagnostics is often hampered by the requirement for a dedicated biosafety level 3 laboratory (BSL-3), the focus of this study is on biosafety aspects to facilitate safe testing also under BSL-2 conditions. To this aim, it was analyzed whether OPXV, which can be handled under BSL-2 conditions, could be used as less virulent alternatives to MPXV. Furthermore, an inactivation method was established to remove up to five log-steps of infectious virus particles from viraemic sera without compromising antibody detection. The results show that immunological cross-reactivity between OPXV provides an opportunity for the interchangeable usage of different OPXV species in serological assays, enabling MPXV serology outside of BSL-3 facilities.

Comparative virome analysis of individual shedding routes of Miniopterus phillipsi bats inhabiting the Wavul Galge cave, Sri Lanka.

Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the full picture of the bat viromes is not explored yet. In this study, the virome composition of Miniopterus phillipsi bats (formerly known as Miniopterus fuliginosus bats in Sri Lanka) inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible excretion routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions, whereby a special focus was set on RNA viruses that are typically associated with bats. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples and further sequences related to Astroviridae in urine samples. No viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several novel viruses represent first reports of these pathogens from bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. phillipsi bats. Based on phylogenetics, the identified viruses are closely related to bat-associated viruses with comparably low estimation of human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

Extensive ITR expansion of the 2022 Mpox virus genome through gene duplication and gene loss.

Poxviruses are known to evolve slower than RNA viruses with only 1-2 mutations/genome/year. Rather than single mutations, rearrangements such as gene gain and loss, which have been discussed as a possible driver for host adaption, were described in poxviruses. In 2022 and 2023 the world is being challenged by the largest global outbreak so far of Mpox virus, and the virus seems to have established itself in the human community for an extended period of time. Here, we report five Mpox virus genomes from Germany with extensive gene duplication and loss, leading to the expansion of the ITR regions from 6400 to up to 24,600 bp. We describe duplications of up to 18,200 bp to the opposed genome end, and deletions at the site of insertion of up to 16,900 bp. Deletions and duplications of genes with functions of supposed immune modulation, virulence and host adaption as B19R, B21R, B22R and D10L are described. In summary, we highlight the need for monitoring rearrangements of the Mpox virus genome rather than for monitoring single mutations only.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents.

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Effectiveness of vaccines in preventing hospitalization due to COVID-19: A multicenter hospital-based case-control study, Germany, June 2021 to January 2022.

We included 852 patients in a prospectively recruiting multicenter matched case-control study in Germany to assess vaccine effectiveness (VE) in preventing COVID-19-associated hospitalization during the Delta-variant dominance. The two-dose VE was 89 % (95 % CI 84-93 %) overall, 79 % in patients with more than two comorbidities and 77 % in adults aged 60-75 years. A third dose increased the VE to more than 93 % in all patient-subgroups.

First Complete Cytochrome B Sequences and Molecular Taxonomy of Bat Species from Sri Lanka.

This is the first report on the molecular identification and phylogeny of the Rousettus leschenaultii Desmarest, 1810, Rhinolophus rouxii Temminck, 1835, Hipposideros speoris Schneider, 1800, Hipposideros lankadiva Kelaart, 1850, and Miniopterus fuliginosus Kuhl, 1817, bat species in Sri Lanka, inferred from analyses by mitochondrially encoded cytochrome b gene sequences. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even within the same species, the acoustic properties of echolocation calls and morphological features such as fur color could vary in different populations. Therefore, we have used molecular taxonomy for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. The bats were caught using a hand net, and saliva samples were collected non-invasively from each bat by using a sterile oral swab. Nucleic acids were extracted from the oral swab samples, and mitochondrial DNA was amplified by using primers targeting the mitochondrially encoded cytochrome b gene. This study reports the first molecular evidence for the identification of five bat species in Sri Lanka. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats which will contribute significantly to the investigation of potentially zoonotic bat viruses.

Paramyxovirus Diversity within One Population of Miniopterus fuliginosus Bats in Sri Lanka.

Bats are known as typical reservoirs for a number of viruses, including viruses of the family Paramyxoviridae. Representatives of the subfamily Orthoparamyxovirinae are distributed worldwide and can cause mild to fatal diseases when infecting humans. The research on Paramyxoviruses (PMVs) from different bat hosts all over the world aims to understand the diversity, evolution and distribution of these viruses and to assess their zoonotic potential. A high number of yet unclassified PMVs from bats are recorded. In our study, we investigated bat species from the families Rhinolophidae, Hipposiderae, Pteropodidae and Miniopteridae that are roosting sympatrically in the Wavul Galge cave (Koslanda, Sri Lanka). The sampling at three time points (March and July 2018; January 2019) and screening for PMVs with a generic PCR show the presence of different novel PMVs in 10 urine samples collected from Miniopterus fuliginosus. Sequence analysis revealed a high similarity of the novel strains among each other and to other unclassified PMVs collected from Miniopterus bats. In this study, we present the first detection of PMVs in Sri Lanka and the presence of PMVs in the bat species M. fuliginosus for the first time.

Full Genome of batCoV/MinFul/2018/SriLanka, a Novel Alpha-Coronavirus Detected in Miniopterus fuliginosus, Sri Lanka.

Coronaviruses (CoV) are divided into the genera α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and ß-CoVs are solely capable of causing infections in humans, resulting in mild to severe respiratory symptoms. Bats have been identified as natural reservoir hosts for CoVs belonging to these two genera. Consequently, research on bat populations, CoV prevalence in bats and genetic characterization of bat CoVs is of special interest to investigate the potential transmission risks. We present the genome sequence of a novel α-CoV strain detected in rectal swab samples of Miniopterus fuliginosus bats from a colony in the Wavul Galge cave (Koslanda, Sri Lanka). The novel strain is highly similar to Miniopterus bat coronavirus 1, an α-CoV located in the subgenus of Minunacoviruses. Phylogenetic reconstruction revealed a high identity of the novel strain to other α-CoVs derived from Miniopterus bats, while human-pathogenic α-CoV strains like HCoV-229E and HCoV-NL63 were more distantly related. Comparison with selected bat-related and human-pathogenic strains of the ß-CoV genus showed low identities of ~40%. Analyses of the different genes on nucleotide and amino acid level revealed that the non-structural ORF1a/1b are more conserved among α-CoVs and ß-CoVs, while there are higher variations in the structural proteins known to be important for host specificity. The novel strain was named batCoV/MinFul/2018/SriLanka and had a prevalence of 50% (66/130) in rectal swab samples and 58% (61/104) in feces samples that were collected from Miniopterus bats in Wavul Galge cave. Based on the differences between strain batCoV/MinFul/2018/SriLanka and human-pathogenic α-CoVs and ß-CoVs, we conclude that there is a rather low transmission risk to humans. Further studies in the Wavul Galge cave and at other locations in Sri Lanka will give more detailed information about the prevalence of this virus.

Detection of Alpha- and Betacoronaviruses in Miniopterus fuliginosus and Rousettus leschenaultii, two species of Sri Lankan Bats.

Bats are known to be potential reservoirs of numerous human-pathogenic viruses. They have been identified as natural hosts for coronaviruses, causing Severe Acute Respiratory Syndrome (SARS) in humans. Since the emergence of SARS-CoV-2 in 2019 interest in the prevalence of coronaviruses in bats was newly raised. In this study we investigated different bat species living in a sympatric colony in the Wavul Galge cave (Koslanda, Sri Lanka). In three field sessions (in 2018 and 2019), 395 bats were captured (Miniopterus, Rousettus, Hipposideros and Rhinolophus spp.) and either rectal swabs or fecal samples were collected. From these overall 396 rectal swab and fecal samples, the screening for coronaviruses with nested PCR resulted in 33 positive samples, 31 of which originated from Miniopterus fuliginosus and two from Rousettus leschenaultii. Sanger sequencing and phylogenetic analysis of the obtained 384-nt fragment of the RNA-dependent RNA polymerase revealed that the examined M. fuliginosus bats excrete alphacoronaviruses and the examined R. leschenaultii bats excrete betacoronaviruses. Despite the sympatric roosting habitat, the coronaviruses showed host specificity and seemed to be limited to one species. Our results represent an important basis to better understand the prevalence of coronaviruses in Sri Lankan bats and may provide a basis for pursuing studies on particular bat species of interest.

Update on Potentially Zoonotic Viruses of European Bats.

Bats have been increasingly gaining attention as potential reservoir hosts of some of the most virulent viruses known. Numerous review articles summarize bats as potential reservoir hosts of human-pathogenic zoonotic viruses. For European bats, just one review article is available that we published in 2014. The present review provides an update on the earlier article and summarizes the most important viruses found in European bats and their possible implications for Public Health. We identify the research gaps and recommend monitoring of these viruses.

The virome of German bats: comparing virus discovery approaches.

Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies.

First detection of bat-borne Issyk-Kul virus in Europe.

Bats have been gaining attention as potential reservoir hosts of numerous viruses pathogenic to animals and man. Issyk-Kul virus, a member of the family Nairoviridae, was first isolated in the 1970s from vespertilionid bats in Central Asia. Issyk-Kul virus has been described as human-pathogenic virus, causing febrile outbreaks in humans with headaches, myalgia and nausea. Here we describe the detection of a novel strain of Issyk-Kul virus from Eptesicus nilssonii in Germany. This finding indicates for the first time the prevalence of these zoonotic viruses in Europe.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Zwiesel bat banyangvirus, a potentially zoonotic Huaiyangshan banyangvirus (Formerly known as SFTS)-like banyangvirus in Northern bats from Germany.

Bats are reservoir hosts for several emerging and re-emerging viral pathogens causing morbidity and mortality in wildlife, animal stocks and humans. Various viruses within the family Phenuiviridae have been detected in bats, including the highly pathogenic Rift Valley fever virus and Malsoor virus, a novel Banyangvirus with close genetic relation to Huaiyangshan banyangvirus (BHAV)(former known as Severe fever with thrombocytopenia syndrome virus, SFTSV) and Heartland virus (HRTV), both of which have caused severe disease with fatal casualties in humans. In this study we present the whole genome of a novel Banyangvirus, named Zwiesel bat banyangvirus, revealed through deep sequencing of the Eptesicus nilssonii bat virome. The detection of the novel bat banyangvirus, which is in close phylogenetic relationship with the pathogenic HRTV and BHAV, underlines the possible impact of emerging phenuiviruses on public health.

PAIPline: pathogen identification in metagenomic and clinical next generation sequencing samples.

Motivation: Next generation sequencing (NGS) has provided researchers with a powerful tool to characterize metagenomic and clinical samples in research and diagnostic settings. NGS allows an open view into samples useful for pathogen detection in an unbiased fashion and without prior hypothesis about possible causative agents. However, NGS datasets for pathogen detection come with different obstacles, such as a very unfavorable ratio of pathogen to host reads. Alongside often appearing false positives and irrelevant organisms, such as contaminants, tools are often challenged by samples with low pathogen loads and might not report organisms present below a certain threshold. Furthermore, some metagenomic profiling tools are only focused on one particular set of pathogens, for example bacteria. Results: We present PAIPline, a bioinformatics pipeline specifically designed to address problems associated with detecting pathogens in diagnostic samples. PAIPline particularly focuses on userfriendliness and encapsulates all necessary steps from preprocessing to resolution of ambiguous reads and filtering up to visualization in a single tool. In contrast to existing tools, PAIPline is more specific while maintaining sensitivity. This is shown in a comparative evaluation where PAIPline was benchmarked along other well-known metagenomic profiling tools on previously published well-characterized datasets. Additionally, as part of an international cooperation project, PAIPline was applied to an outbreak sample of hemorrhagic fevers of then unknown etiology. The presented results show that PAIPline can serve as a robust, reliable, user-friendly, adaptable and generalizable stand-alone software for diagnostics from NGS samples and as a stepping stone for further downstream analyses. Availability and implementation: PAIPline is freely available under https://gitlab.com/rki_bioinformatics/paipline.

Tissue-Based Universal Virus Detection (TUViD-VM) Protocol for Viral Metagenomics.

The protocol for tissue-based universal virus detection (TUViD-VM) was developed to allow for the detection of yet unknown viruses from infected tissues. Tissues are a challenging source when it comes to virus detection. The very rapid degradation rate of tissue and the high level of host nucleic acids are the biggest challenges when examining tissues. However, to overcome these challenges, we had to decrease the amount of host nucleic acids rigorously in order to increase the amount of viral nucleic acids. We compared modern and common virological approaches to find the optimal conditions. The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other approaches tested. The TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source. The protocol has been widely used to detect viruses in different tissues with great success. Here we provide the bench-top protocol version of the TUViD-VM protocol.

Hervey virus: Study on co-circulation with Henipaviruses in Pteropid bats within their distribution range from Australia to Africa.

In 2011, an unusually large number of independent Hendra virus outbreaks were recorded on horse properties in Queensland and New South Wales, Australia. Urine from bat colonies adjacent to the outbreak sites were sampled and screened for Hendra and other viruses. Several novel paramyxoviruses were also isolated at different locations. Here one of the novel viruses, named Hervey virus (HerPV), is fully characterized by genome sequencing, annotation, phylogeny and in vitro host range, and its serological cross-reactivity and neutralization patterns are examined. HerPV may have ecological and spatial and temporal patterns similar to Hendra virus and could serve as a sentinel virus for the surveillance of this highly pathogenic virus. The suitability of HerPV as potential sentinel virus is further assessed by determining the serological prevalence of HerPV antibodies in fruit-eating bats from Australia, Indonesia, Papua New Guinea, Tanzania and the Gulf of Guinea, indicating the presence of similar viruses in regions beyond the Australian border.

Use of RNALater® Preservation for Virome Sequencing in Outbreak Settings.

Outbreaks of infectious diseases may occur in animal and human populations; this underlines the need for suitable One Health approaches. During outbreak situations, straightforward identification of etiological agents is indispensable for taking countermeasures. A recently published protocol for metagenomic virus detection in clinical specimens (TUViD-VM) was developed for snap-frozen tissues which can be challenging to obtain. Here, we describe the use of RNALater® -treated tissue at ambient temperatures for virome sequencing. This study demonstrates that samples stored in RNALater® buffer yield similar results to those stored snap-frozen.