Current understanding of an Emerging Coronavirus using in silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

Novel coronavirus (nCoV) namely “SARS-CoV-2” is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the “SARS-CoV-2” although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among “SARS-CoV-2” and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that “SARS-CoV-2” has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

Molecular Characterization of Novel Cucurbit Aphid Borne Yellows Virus Strains Infecting Squash and Watermelon in India

The pathogen responsible for yellowing and downward rolling of leaves of squash and watermelon plants from Uttar Pradesh state, India, was identified as probably strains of Cucurbit aphid-borne yellows virus (CABYV) through RT-PCR using universal Polerovirus primers followed by sequencing. The full-length genome sequences of an isolate from squash (POL-SQ - 5650 nt) and one from watermelon (POL-WM - 5647nt) were determined by sequencing the products from RT-PCR with six sets of primers with overlapping products. Sequence comparison and phylogenetic analysis showed that these isolates had closest identity with a recombinant strain obtained between CABYV and Melon aphid-borne yellows virus (MABYV) reported from Taiwan infecting Luffa aegyptiaca (CABYV-R-TW82) rather than other Asian, American, or European isolates. The deduced amino acid sequences of the P0, P1 and P1-P2 proteins showed >10% variation, whereas the P3, P4 and P3-P5 proteins showed <10% variation when compared to the corresponding proteins of other strains of CABYV worldwide. Thus, according to the Polerovirus species demarcation threshold, these new sequences should be regarded as representing strains of a novel previously undescribed Polerovirus species. However, based on their sequence similarity and phylogenetic grouping with the recombinant strain from Taiwan we suggest these sequences represent recombinant strains of CABYV. These are the first full-length genome sequences for CABYV strains from India and this study adds watermelon as host for CABYV in India.

A one health approach to investigating an outbreak of alimentary tick-borne encephalitis in a non-endemic area in France (Ain, Eastern France): a longitudinal serological study in livestock, detection in ticks, and the first TBE virus isolation and molecular characterization

Tick borne encephalitis virus geographic range and human incidence is increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of TBE in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurized raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and to witness in real time the seroconversion of recently exposed individuals and the excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for as a source of dietary contamination in France, obtained its full-length genome sequence, and found that it does not cluster very closely neither with the isolate circulating in Alsace nor with any other isolate within the European lineage. TBEV is now a notifiable human disease in France, which should facilitate surveillance of TBEV incidence and distribution throughout France.

Genomic Evaluation of the Genus Coltivirus Indicates Genetic Diversity among Colorado Tick Fever Virus Strains and Demarcation of a New Species

The type species of the genus Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.

Genetic Characterization and Recombinant History of a Novel Emerging HIV-1 Second-Generation Circulating Recombinant Form (CRF120_0107) Identified Among Sexually Transmitted Infections in Shenzhen, China

Under the background of the main epidemic HIV strains (CRF01_AE and CRF07_BC) co-circulation in China, more HIV second-generation recombinant (SGR) strains with CRF01_AE and CRF07_BC as the backbone are emerging. In this study, we analyzed the characteristics and evolutionary history of a newly emerging HIV-1 CRF120_0107 composed of CRF01_AE and CRF07_BC based on the near full-length genome (NFLG) in Shenzhen, Guangdong Province, China. NFLG phylogenetic analysis revealed that these sequences formed a distinct monophyletic branch with a high bootstrap value (>90%), distantly related to all known HIV-1 genotypes. Recombination analysis showed that CRF120_0107 was composed of the predominant HIV-1 strains in China: CRF01_AE and CRF07_BC. Further subregional phylogenetic analysis was performed that possible parental lineages of CRF07_BC segments (Ⅰ, Ⅲ, and Ⅴ) belonged to the CRF07_BC men who have sex with men cluster (MSM cluster), other CRF01_AE segments also mainly belonged to MSM Cluster (such as CRF01_AE Cluster 5). Bayesian analysis results inferred that CRF120_0107 placed its emergence in Shenzhen approximately between 2009-2011. The appearance of CRF120_0107 further highlights that more and more HIV-1 SGR strains containing CRF01_AE and CRF07_BC will be more generated frequently and will most likely be more conducive to accelerating the spread of HIV in China. This highlighted it is necessary to monitor MSM high-risk individuals with HIV-1 CRF01_AE and CRF07_BC dual infection to prevent the generation of CRF01_AE/CRF07_BC recombinant strains, thus reducing the possibility of HIV-1 genotype resistance and the complexity of treatment in China.

LinearTurboFold: Linear-time global prediction of conserved structures for RNA homologs with applications to SARS-CoV-2

The constant emergence of COVID-19 variants reduces the effectiveness of existing vaccines and test kits. Therefore, it is critical to identify conserved structures in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes as potential targets for variant-proof diagnostics and therapeutics. However, the algorithms to predict these conserved structures, which simultaneously fold and align multiple RNA homologs, scale at best cubically with sequence length and are thus infeasible for coronaviruses, which possess the longest genomes (∼30,000 nt) among RNA viruses. As a result, existing efforts on modeling SARS-CoV-2 structures resort to single-sequence folding as well as local folding methods with short window sizes, which inevitably neglect long-range interactions that are crucial in RNA functions. Here we present LinearTurboFold, an efficient algorithm for folding RNA homologs that scales linearly with sequence length, enabling unprecedented global structural analysis on SARS-CoV-2. Surprisingly, on a group of SARS-CoV-2 and SARS-related genomes, LinearTurboFold’s purely in silico prediction not only is close to experimentally guided models for local structures, but also goes far beyond them by capturing the end-to-end pairs between 5′ and 3′ untranslated regions (UTRs) (∼29,800 nt apart) that match perfectly with a purely experimental work. Furthermore, LinearTurboFold identifies undiscovered conserved structures and conserved accessible regions as potential targets for designing efficient and mutation-insensitive small-molecule drugs, antisense oligonucleotides, small interfering RNAs (siRNAs), CRISPR-Cas13 guide RNAs, and RT-PCR primers. LinearTurboFold is a general technique that can also be applied to other RNA viruses and full-length genome studies and will be a useful tool in fighting the current and future pandemics.

Genome-scale molecular and phylogenetic characterization of Middle Point orbiviruses from Australia

Middle Point orbivirus (MPOV) is an Australian arbovirus, belongs to the Yunnan orbivirus species found in China. First detected and reported from Beatrice Hill, Northern Territory (NT), MPOV has to date, only been exclusively reported from the NT, Australia. Whilst genetic characterization of MPOV has been previously described, only restricted to sequence information for segments 2 and 3 coding core protein VP2 and outer capsid protein VP3, respectively. This study presents for the first time nearly full-length genome sequences of MPOV, which represent 24 isolates collected over a span of more than 20 years from 1997 to 2018. Whilst the majority of isolates were sampled at Beatrice Hill, NT where MPOV is most frequently isolated, this report also describes the first two isolations of MPOV from Queensland (QLD), Australia. One of which is the first non-bovine isolate obtained from the mosquito vector Aedes vittiger. We further compared these MPOV sequences with known sequences of the Yunnan orbivirus and other known orbivirus sequences of mosquito origin found in Australia. The phylogenetic analyses indicate the Australian MPOV sequences are more closely related to each other than other known sequences of Yunnan orbivirus. Furthermore, MPOV sequences are closely related to sequences from the Indonesian isolate JKT-8650. The clustering of Australian sequences in the phylogenetic tree suggests the monophyletic lineage of MPOV circulating in Australia. Further, ongoing surveillance is required to assess the existence and prevalence of this or other yet undetected lineages of MPOV and other orbiviruses in Australia.

Phylogenetic and full-length genome mutation analysis of SARS-CoV-2 in Indonesia prior to COVID-19 vaccination program in 2021

Background Indonesia has started the big project of COVID-19 vaccination program since 13 January 2021 by employing the first shot of vaccine to the President of Indonesia as the outbreak and rapid transmission of COVID-19 have endangered not only Indonesian but the global health and economy. This study aimed to investigate the full-length genome mutation analysis of 166 Indonesian SARS-CoV-2 isolates as of 12 January 2021. Results All data of the isolates were extracted from the Global Initiative on Sharing All Influenza Data (GISAID) EpiCoV database. CoVsurver platform was employed to investigate the full-length genome mutation analysis of all isolates. This study also focused on the phylogeny analysis in unlocking the mutation of S protein in Indonesian SARS-CoV-2 isolates. WIV04 isolate that was originated from Wuhan, China was used as the virus reference according to the CoVsurver default. The result showed that a full-length genome mutation analysis of 166 Indonesian SARS-CoV-2 isolates was successfully generated. Every single mutation in S protein was described and then visualized by utilizing BioRender platform. Furthermore, it also found that D614G mutation appeared in 103 Indonesian SARS-CoV-2 isolates. Conclusions To sum up, this study helped to observe the spread of COVID-19 transmission. However, it also proposed that the epidemiological surveillance and genomics studies might be improved on COVID-19 pandemic in Indonesia.

Complete Genome Sequence of a Novel Potyvirus Infecting Miscanthus Sinensis (Silver Grass)

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named “miscanthus sinensis mosaic virus” (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post entry quarantine facility following its initial import into Western Australia, Australia. The MsiMV genome encompasses 9604 nucleotides (nt) encoding a 3071 amino acids (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to sorghum mosaic virus (SrMV) with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Identification of CRF66_BF, a New HIV-1 Circulating Recombinant Form of South American Origin

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 110 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n = 4), Spain (n = 3), Paraguay (n = 1), Brazil (n = 1), and Italy (n = 1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17gag, integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF “family” clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.