The Analysis of Oral and Fecal Virome Detects Multiple Novel Emerging Viruses in Snakes

Wild animals are considered reservoirs for emerging and reemerging viruses, such as the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies have reported that bats and ticks harbored variable important pathogenic viruses, some of which could cause potential diseases in humans and livestock, while viruses carried by reptiles were rarely reported. Our study first conducted snakes' virome analysis to establish effective surveillance of potential transboundary emerging diseases. Consequently, Adenoviridae, Circoviridae, Retroviridae, and Parvoviridae were identified in oral samples from Protobothrops mucrosquamatus, Elaphe dione, and Gloydius angusticeps based on sequence similarity to existing viruses. Picornaviridae and Adenoviridae were also identified in fecal samples of Protobothrops mucrosquamatus. Notably, the iflavirus and foamy virus were first reported in Protobothrops mucrosquamatus, enriching the transboundary viral diversity in snakes. Furthermore, phylogenetic analysis revealed that both the novel-identified viruses showed low genetic similarity with previously reported viruses. This study provided a basis for our understanding of microbiome diversity and the surveillance and prevention of emerging and unknown viruses in snakes.

Isolation of a Novel Bat Rhabdovirus with Evidence of Human Exposure in China.

Bats are well-recognized reservoirs of zoonotic viruses. Several spillover events from bats to humans have been reported, causing severe epidemic or endemic diseases including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome-CoV (MERS-CoV), henipaviruses, and filoviruses. In this study, a novel rhabdovirus species, provisionally named Rhinolophus rhabdovirus DPuer (DPRV), was identified from the horseshoe bat (Rhinolophus affinis) in Yunnan province, China, using next-generation sequencing. DPRV shedding in the spleen, liver, lung, and intestinal contents of wild bats with high viral loads was detected by real-time quantitative PCR, indicating that DPRV has tropism for multiple host tissues. Furthermore, DPRV can replicate in vitro in multiple mammalian cell lines, including BHK-21, A549, and MA104 cells, with the highest efficiency in hamster kidney cell line BHK-21, suggesting infectivity of DPRV in these cell line-derived hosts. Ultrastructure analysis revealed a characteristic bullet-shaped morphology and tightly clustered distribution of DPRV particles in the intracellular space. DPRV replicated efficiently in suckling mouse brains and caused death of suckling mice; death rates increased with passaging of DPRV in suckling mice. Moreover, 421 serum samples were collected from individuals who lived near the bat collection site and had fever symptoms within 1 year. DPRV-specific antibodies were detected in 20 (4.75%) human serum samples by indirect immunofluorescence assay. Furthermore, 10 (2.38%) serum samples were DPRV positive according to plaque reduction neutralization assay, which revealed potential transmission of DPRV from bats to humans and highlighted the potential public health risk. Potential vector association with DPRV was not found with negative viral RNA in bloodsucking arthropods. IMPORTANCE We identified a novel rhabdovirus from the horseshoe bat (Rhinolophus thomasi) in China with probable infectivity in humans. DPRV was isolated in vitro from several mammalian cell lines, indicating wide host tropism, excluding bats, of DPRV. DPRV replicated in the brains of suckling mice, and the death rate of suckling mice increased with passaging of DPRV in vivo. Serological tests indicated the possible infectivity of DPRV in humans and the potential transmission to humans. The present findings provide preliminary evidence for the potential risk of DPRV to public health. Additional studies with active surveillance are needed to address interspecies transmission and determine the pathogenicity of DPRV in humans.

A multicenter study on molecular epidemiology of rhinovirus isolated from children with community acquired pneumonia in China during 2017-2019.

Rhinoviruses (RVs) are a major pathogen of community acquired pneumonia in children. To investigate the prevalence and genetic characteristics of RVs in China, we performed a molecular epidemiological study during 2017-2019 in community acquired pneumonia (CAP) in pediatric patients. In this multicenter study, 109 RV-A, 20 RV-B and 80 RV-C were identified. Among them, RV-A12, RV-A101, RV-A78, RV-A49, RV-A22, RV-B52, RV-C2, RV-C53 and RV-C5 were the common genotypes in the study. A total of 23 complete genome of RVs including 4 RV-A, 1 RV-B and 18 RV-C were obtained. Furthermore, in the RV-C isolates, one RV-C5 and five RV-C53 genotypes were found, which have a limited number in the GenBank. Phylogenetic analysis of the complete genome showed that most of the RVs isolated in the study have high nucleotide sequence identities (>95%) compared with the corresponding reference sequence in the GenBank. In RV-A9, RV-A28, RV-A61 and RV-B52, amino acid mutations were found in the potential neutralizing immunogenic (Nim) sites (Nim-1a and Nim-1b) of the VP1. In RV-B52, one of RV-C2 and RV-C5 isolates, amino acid mutations were found in the P1a peptide of the VP1. However, no recombination events were found in the study. In conclusion, RV-A was the predominant specie of RVs followed by RV-C in the study. The complete genomes of one RV-C5 and five RV-C53 genotypes were obtained which have a limited number sequence in the GenBank. High nucleotide sequence identities (>95%) were found among the complete genome obtained in the study and the corresponding reference sequence in the GenBank. Amino acid mutations were found in the potential Nim-1a, Nim-1b sites and P1a peptide region of the VP1 in parts of RVs.

Complete genome sequencing of SARS-CoV-2 strains: A pilot survey in Palestine reveals spike mutation H245N.

OBJECTIVES: SARS-CoV-2, severe respiratory syndrome coronavirus-2, is an RNA virus that emerged from China sweeping the globe in the form of a pandemic that became an international public health concern. This pilot study aimed to describe the genetic variation and molecular epidemiology of SARS-CoV-2 in Palestine in fall 2020. RESULTS: To achieve these aims, whole genome sequencing of SARS-CoV-2, phylogenetic analysis, haplotype networking and genetic diversity analysis were performed. These analyses revealed a unique spike mutation H245N that has never been reported before. The phylogenetic analysis depicted that three clusters existed in Palestinian SARS-CoV-2 genome sequences, in which cluster-I comprised the majority of clusters by 90%. Congruently, the haplotype network analysis depicted the same three clusters with a total of 39 haplotypes. The genetic diversity analysis showed that Cluster-I is highly diverse as confirmed by statistically significant mutation rate indices, Tajima's D and Fu-Li's-F tests (- 2.11 and 2.74, respectively), highest number of mutations (Eta = 120), highest number of haplotypes (h = 17), and highest average number of nucleotide differences between any two sequences (S = 118). The study confirmed the high genetic diversity among the Palestinian of SARS-CoV-2 which possessed high number of mutations including one which was reported for the first time.

New Progress of Glycosylation in Coronaviruses

The coronaviruses (CoVs), which are a family of positive-strand RNA viruses, infect the mammals and birds. Seven CoVs are responsible for human-to-human transmission, especially the SARS-CoV-2, thereby posing a daunting challenge to global public health security. As the most common modification in viral glycoproteins, glycosylation plays the crucial role in host recognition, immunity avoidance, virus replication, assembly and transmission. In this review, we summarized and discussed the latest studies about glycosylation in coronaviridae members. Focused on the spike protein, nearly one hundred of N/O-glycosyltion sites have been reported. The N-glycans from spike protein are dominated by the high-mannose and complex-type, while the O-glycosylation is rather complicated. Significantly, it is known that the viral glycosylation depend on host cells, thus the glycan pattern of the produced recombinant viral glycoproteins might be different from that of native viral proteins, which represent a crucial determinant for vaccine design. The latest results based on bioinformatics, biochip, mass spectrography and genetic technology facilitate the overall perspective for glycosylation researching in CoVs. By summarizing the distribution of glycosylation sites, the structure of glycans, the biological functions and the research technologies, this review will help promote diagnosis, treatment and vaccine development related to coronaviruses.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

SARS-CoV-2 Circulation, Guinea, March 2020-July 2021.

This overview of severe acute respiratory syndrome coronavirus 2 circulation over 1.5 years in Guinea demonstrates that virus clades and variants of interest and concern were progressively introduced, mostly by travellers through Conakry, before spreading through the country. Sequencing is key to following virus evolution and establishing efficient control strategies.

Global SNP analysis of 11,183 SARS-CoV-2 strains reveals high genetic diversity.

Since first identified in December of 2019, COVID-19 has been quickly spreading to the world in few months and COVID-19 cases are still undergoing rapid surge in most countries worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adapts and evolves rapidly in nature. With the availability of 16,092 SARS-CoV-2 full genomes in GISAID as of 13 May, we removed the poor-quality genomes and performed mutational profiling analysis for the remaining 11,183 viral genomes. Global analysis of all sequences identified all single nucleotide polymorphisms (SNPs) across the whole genome and critical SNPs with high mutation frequency that contributes to five-clade classification of global strains. A total of 119 SNPs were found with 74 non-synonymous mutations, 43 synonymous mutations and two mutations in intergenic regions. Analysis of geographic pattern of mutational profiling for the whole genome reveals differences between each continent. A transition mutation from C to T represents the most mutation types across the genome, suggesting rapid evolution and adaptation of the virus in host. Amino acid (AA) deletions and insertions found across the genome results in changes in viral protein length and potential function alteration. Mutational profiling for each gene was analysed, and results show that nucleocapsid gene demonstrates the highest mutational frequency, followed by Nsp2, Nsp3 and Spike gene. We further focused on non-synonymous mutational distributions on four key viral proteins, spike with 75 mutations, RNA-dependent-RNA-polymerase with 41 mutations, 3C-like protease with 22 mutations and Papain-like protease with 10 mutations. Results show that non-synonymous mutations on critical sites of these four proteins pose great challenge for development of anti-viral drugs and other countering measures. Overall, this study provides more understanding of genetic diversity/variability of SARS-CoV-2 and insights for development of anti-viral therapeutics.

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Molecular characterization, phylogenetic and variation analyses of SARS-CoV-2 strains in Turkey.

Aims: We present the sequence and single-nucleotide polymorphism (SNP) analysis for 47 complete genomes for SARS-CoV-2 isolates on Turkish patients. Methods: The Illumina MiSeq platform was used for sequencing the libraries. The SNPs were detected by using Genome Analysis Toolkit - HaplotypeCaller v.3.8.0 and were inspected on GenomeBrowse v2.1.2. Results: All viral genome sequences of our isolates were located in lineage B under the different clusters, such as B.1 (n = 3), B.1.1 (n = 28) and B.1.9 (n = 16). According to the Global Initiative on Sharing All Influenza Data nomenclature, all of our complete genomes were placed in G, GR and GH clades. In our study, 549 total and 53 unique SNPs were detected. Conclusion: The results indicate that the SARS-CoV-2 sequences of our isolates have great similarity with all Turkish and European sequences.

Circulating SARS-CoV-2 variants in Italy, October 2020-March 2021.

A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.

Clinical features, isolation, and complete genome sequence of severe acute respiratory syndrome coronavirus 2 from the first two patients in Vietnam.

In January 2020, we identified two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in a familial cluster with one person coming from Wuhan, China. The complete genome sequences of two SARS-CoV-2 strains isolated from these patients were identical and 99.98% similar to strains isolated in Wuhan. This is genetically suggestive of human-to-human transmission of SARS-CoV-2 and indicates Wuhan as the most plausible origin of the early outbreak in Vietnam. The younger patient had a mild upper respiratory illness and a brief viral shedding, whereas the elderly with multi-morbidity had pneumonia, prolonged viral shedding, and residual lung damage. The evidence of nonsynonymous substitutions in the ORF1ab region of the viral sequence warrants further studies.