Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples.

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since most of these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low- and middle-income countries. In order to develop an inexpensive SARS-CoV-2 detection protocol using available resources we evaluated the SYBR Green based detection of SARS-CoV-2 to establish a suitable assay. To do so, we adapted one of the WHO recommended TaqMan-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results indicate that SYBR-Green detection of ORF1b-nsp14 target represents a reliable cost-effective alternative to increase the testing capacity.

Evidence of increasing diversification of emerging Severe Acute Respiratory Syndrome Coronavirus 2 strains.

On 30th January 2020, an outbreak of atypical pneumonia caused by a novel betacoronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a public health emergency of international concern by the World Health Organization. For this reason, a detailed evolutionary analysis of SARS-CoV-2 strains currently circulating in different geographic regions of the world was performed. A compositional analysis as well as a Bayesian coalescent analysis of complete genome sequences of SARS-CoV-2 strains recently isolated in Europe, North America, South America, and Asia was performed. The results of these studies revealed a diversification of SARS-CoV-2 strains in three different genetic clades. Co-circulation of different clades in different countries, as well as different genetic lineages within different clades were observed. The time of the most recent common ancestor was established to be around 1st November 2019. A mean rate of evolution of 6.57 × 10-4 substitutions per site per year was found. A significant migration rate per genetic lineage per year from Europe to South America was also observed. The results of these studies revealed an increasing diversification of SARS-CoV-2 strains. High evolutionary rates and fast population growth characterizes the population dynamics of SARS-CoV-2 strains.

An evolutionary insight into emerging Ebolavirus strains isolated in Africa.

On July 19, 2019, the World Health Organization declared the current Ebolavirus (EBOV) outbreak in Congo Democratic Republic (COD) a public health emergency of international concern. To address the potential threat of EBOV evolution outpacing antibody treatment and vaccine efforts, a detailed evolutionary analysis of EBOV strains circulating in different African countries was performed. Genome composition of EBOV strains was studied using multivariate statistical analysis. To investigate the patterns of evolution of EBOV strains, a Bayesian Markov Chain Monte Carlo approach was used. Two different genetic lineages, with a distinct genome composition gave rise to the recent EBOV outbreaks in central and western Africa. Strains isolated in COD in 2018 fall into two different genetic clusters, according to their geographical location of isolation. Different amino acid substitutions among strains from these two clusters have been found, particularly in NP, GP, and L proteins. Significant differences in codon and amino acid usage among clusters were found. Strains isolated in COD in 2018 belong to two distinct genetic clusters, with distinct codon and amino acid usage. Geographical diversity plays an important role in shaping the molecular evolution of EBOV populations.

Evolution of naturally occurring 5'non-coding region variants of Hepatitis C virus in human populations of the South American region.

BACKGROUND: Hepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. Previous and recent studies have suggested a diversification of type 1 HCV in the South American region. The degree of genetic variation among HCV strains circulating in Bolivia and Colombia is currently unknown. In order to get insight into these matters, we performed a phylogenetic analysis of HCV 5' non-coding region (5'NCR) sequences from strains isolated in Bolivia, Colombia and Uruguay, as well as available comparable sequences of HCV strains isolated in South America. METHODS: Phylogenetic tree analysis was performed using the neighbor-joining method under a matrix of genetic distances established under the Kimura-two parameter model. Signature pattern analysis, which identifies particular sites in nucleic acid alignments of variable sequences that are distinctly representative relative to a background set, was performed using the method of Korber & Myers, as implemented in the VESPA program. Prediction of RNA secondary structures was done by the method of Zuker & Turner, as implemented in the mfold program. RESULTS: Phylogenetic tree analysis of HCV strains isolated in the South American region revealed the presence of a distinct genetic lineage inside genotype 1. Signature pattern analysis revealed that the presence of this lineage is consistent with the presence of a sequence signature in the 5'NCR of HCV strains isolated in South America. Comparisons of these results with the ones found for Europe or North America revealed that this sequence signature is characteristic of the South American region. CONCLUSION: Phylogentic analysis revealed the presence of a sequence signature in the 5'NCR of type 1 HCV strains isolated in South America. This signature is frequent enough in type 1 HCV populations circulating South America to be detected in a phylogenetic tree analysis as a distinct type 1 sub-population. The coexistence of distinct type 1 HCV subpopulations is consistent with quasispecies dynamics, and suggests that multiple coexisting subpopulations may allow the virus to adapt to its human host populations.