Emerging SARS-CoV-2 Genotypes Show Different Replication Patterns in Human Pulmonary and Intestinal Epithelial Cells

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype–cell type pair for each assay.

Critical Role of the Subways in the Initial Spread of SARS-CoV-2 in New York City

We studied the possible role of the subways in the spread of SARS-CoV-2 in New York City during late February and March 2020. Data on cases and hospitalizations, along with phylogenetic analyses of viral isolates, demonstrate rapid community transmission throughout all five boroughs within days. The near collapse of subway ridership during the second week of March was followed within 1–2 weeks by the flattening of COVID-19 incidence curve. We observed persistently high entry into stations located along the subway line serving a principal hotspot of infection in Queens. We used smartphone tracking data to estimate the volume of subway visits originating from each zip code tabulation area (ZCTA). Across ZCTAs, the estimated volume of subway visits on March 16 was strongly predictive of subsequent COVID-19 incidence during April 1–8. In a spatial analysis, we distinguished between the conventional notion of geographic contiguity and a novel notion of contiguity along subway lines. We found that the March 16 subway-visit volume in subway-contiguous ZCTAs had an increasing effect on COVID-19 incidence during April 1–8 as we enlarged the radius of influence up to 5 connected subway stops. By contrast, the March 31 cumulative incidence of COVID-19 in geographically-contiguous ZCTAs had an increasing effect on subsequent COVID-19 incidence as we expanded the radius up to three connected ZCTAs. The combined evidence points to the initial citywide dissemination of SARS-CoV-2 via a subway-based network, followed by percolation of new infections within local hotspots.

Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

Investigations on Vector-Borne and Aerosol Transmission Potential of Kaeng Khoi Virus in Cave-Dwelling Wrinkle-Lipped Free-Tailed Bats (Chaerephon plicatus) in Thailand

Kaeng Khoi virus (KKV; Order: Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus), is an endemic viral infection of the wrinkle-lipped free-tailed bat (Chaerephon plicatus; also known as Tadarida plicata plicata). Viral isolates from bat bugs (Family: Cimicidae) suggest vector-borne transmission, but in general little is known about the ecology of KKV and seroprevalence in the local human and animal populations. Transmission studies and a serosurvey were carried out in Kaeng Khoi cave, Saraburi province, Thailand, during 1973–1974. Experimental transmission studies were performed with bat bugs captured within the cave to determine the potential for vector-borne transmission, and sentinel laboratory mice placed inside arthropod-proof cages within the cave to assess the potential for aerosolized transmission. Antibodies to KKV were detected in roof rats (Rattus rattus) inhabiting the cave, in dogs living in the valley, and in humans. Freshly collected cimicids were positive for KKV, but the virus did not replicate in laboratory-inoculated bugs. Sentinel mice placed in Kaeng Khoi cave in open cages consistently became infected with KKV, as determined by the development of neutralizing antibodies. Mice placed in arthropod-proof cages also developed antibodies, indicating the possibility of airborne transmission of KKV.

Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017

Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.

Assessment of inter-laboratory differences in SARS-CoV-2 consensus genome assemblies between public health laboratories in Australia

Whole-genome sequencing of viral isolates is critical for informing transmission patterns and ongoing evolution of pathogens, especially during a pandemic. However, when genomes have low variability in the early stages of a pandemic, the impact of technical and/or sequencing errors increases. We quantitatively assessed inter-laboratory differences in consensus genome assemblies of 72 matched SARS-CoV-2-positive specimens sequenced at different laboratories in Sydney, Australia. Raw sequence data were assembled using two different bioinformatics pipelines in parallel, and resulting consensus genomes were compared to detect laboratory-specific differences. Matched genome sequences were predominantly concordant, with a median pairwise identity of 99.997%. Identified differences were predominantly driven by ambiguous site content. Ignoring these produced differences in only 2.3% (5/216) of pairwise comparisons, each differing by a single nucleotide. Matched samples were assigned the same Pango lineage in 98.2% (212/216) of pairwise comparisons, and were mostly assigned to the same phylogenetic clade. However, epidemiological inference based only on single nucleotide variant distances may lead to significant differences in the number of defined clusters if variant allele frequency thresholds for consensus genome generation differ between laboratories. These results underscore the need for a unified, best-practices approach to bioinformatics between laboratories working on a common outbreak problem.

Begomovirus Caboniensis: a New Bipartite Begomovirus Isolated From Cnidoscolus Urens in Brazil

A novel bipartite begomovirus infecting Cnidoscolus urens (Euphorbiaceae) from Pernambuco State, Brazil has been characterized. The complete DNA-A (2657 to 2692 nt) and DNA-B (2622 nt) components of the viral isolates showed a typical genome organization of New World bipartite begomoviruses. DNA-A of the isolates had the highest percentage of nucleotide identity (88.6–88.9%) with the Cnidoscolus mosaic leaf deformation virus (NC_038982). Based on the current classification criteria for the genus Begomovirus, a new member infecting C. urens was reported, and the name Begomovirus caboniensis was proposed for these viruses, adopting the standardized binomial system.

Importance of mutations in amino acid 484 of the Spike protein of SARS-CoV-2: rapid detection by restriction enzyme analysis

Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 484 of the Spike protein are particularly important and associated with some of these variants: E484K or E484Q. These mutations have been associated with evasion to neutralizing antibodies. Restriction enzyme analysis is proposed as a rapid method to detect these mutations. A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. E484K, present in some VOCs, has emerged in several other lineages and is frequently found in recent viral isolates. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, and MseI. The use of these two enzymes allows the detection of mutations at position 484, and to differentiate between these three conditions: non-mutated, and the presence of E484K or E484Q. A 100% correlation was observed with sequencing results. The proposed methodology, which allows for the screening of a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.