Background The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. Aim Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). Methods Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. Results Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. Conclusion The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

Coronaviruses (CoV) are divided into the genera α-CoVs, β-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and β-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 β-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 β-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 β‑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.

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.

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.

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.

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 from Miniopterus fuliginosus bats inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible shedding 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. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Retroviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples, and further sequences related to Astroviridae in urine samples. No further 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 viruses were detected for the first time in bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. fuliginosus bats. Based on phylogenetics, the identified viruses are closer related to bat-associated viruses with comparably low human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

This is the first report on the molecular identification and phylogeny of Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva, Miniopterus fuliginosus bat species in Sri Lanka, inferred from mitochondrially encoded cytochrome b gene sequences. Wellawaya Wavulgalge cave in Sri Lanka is one of the largest sympatric colonies found on the island, occupied by five species of bats. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even in the same species, acoustic properties of echolocation calls and morphological features such as fur colour could vary in different populations. Therefore, we have used molecular techniques for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. Bats were caught using a hand net and saliva samples were collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted from oral swab samples and mitochondrial DNA was amplified using primers targeting the mitochondrially encoded cytochrome b gene. This study identified the bat species recorded in the Wellawaya cave as Rousettus leschenaultii , Rhinolophus rouxii , Hipposideros speoris , Hipposideros lankadiva and Miniopterus fuliginosus . 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 .

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.