Multiplexed Magnetofluorescent Bioplatform for the Sensitive Detection of SARS-CoV-2 Viral RNA without Nucleic Acid Amplification.

Rapid and sensitive detection of SARS-CoV-2 virus genetic material is of paramount importance to mitigate the COVID-19 pandemic outbreak and lower the death toll. Herein, we report the design of a magnetofluorescent bioplatform for the direct and specific detection of the viral RNA of SARS-CoV-2 in the total RNA extracted from nasopharyngeal swabs of COVID-19-positive patients. A higher fluorescence response was achieved using two capture probes tethered to magnetic beads using a biotin/streptavidin linkage, targeting two specific sites in the ORF1a and S genes. Two horseradish peroxidase (HRP)-conjugated reporter sequences, complementary to the loci of the S and N genes, were used to reveal the presence of the viral RNA through the oxidation of o-phenylenediamine to fluorescent 2,3-diaminophenazine. Under optimal conditions, the bioplatform showed high selectivity and sensitivity and was able to detect as low as 0.01 ng of viral RNA (1 × 103 copies/µL) with a linear dynamic range varying from 0.01 to 3.0 ng (1 × 103 to 9 × 107 copies/µL). The bioplatform was also able to discriminate the SARS-CoV-2 RNA from those of other related viruses such as hepatitis C, West Nile, measles, and non-polio viruses. Furthermore, the developed biosensor was validated in 46 clinical samples (36 COVID-19-positive patients and 10 COVID-19-negative subjects, as assessed with the gold standard RT-qPCR method). Both sensitivity and specificity of the developed method reached 100%. Finally, making such a simple and specific method available in the field, at a primary point of care, can better help the detection of SARS-CoV-2 infection in low-resource settings.

Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa.

BACKGROUND: In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide. METHODS: Whole genome sequencing was performed using NGS approach on six SARS. CoV-2 highly positive samples detected during the early phase of the outbreak. RESULTS: Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development. CONCLUSIONS: These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.

Circulation and Molecular Epidemiology of Enteroviruses in Paralyzed, Immunodeficient and Healthy Individuals in Tunisia, a Country with a Polio-Free Status for Decades.

This report is an overview of enterovirus (EV) detection in Tunisian polio-suspected paralytic cases (acute flaccid paralysis (AFP) cases), healthy contacts and patients with primary immunodeficiencies (PID) during an 11-year period. A total of 2735 clinical samples were analyzed for EV isolation and type identification, according to the recommended protocols of the World Health Organization. Three poliovirus (PV) serotypes and 28 different nonpolio enteroviruses (NPEVs) were detected. The NPEV detection rate was 4.3%, 2.8% and 12.4% in AFP cases, healthy contacts and PID patients, respectively. The predominant species was EV-B, and the circulation of viruses from species EV-A was noted since 2011. All PVs detected were of Sabin origin. The PV detection rate was higher in PID patients compared to AFP cases and contacts (6.8%, 1.5% and 1.3% respectively). PV2 was not detected since 2015. Using nucleotide sequencing of the entire VP1 region, 61 strains were characterized as Sabin-like. Among them, six strains of types 1 and 3 PV were identified as pre-vaccine-derived polioviruses (VDPVs). Five type 2 PV, four strains belonging to type 1 PV and two strains belonging to type 3 PV, were classified as iVDPVs. The data presented provide a comprehensive picture of EVs circulating in Tunisia over an 11-year period, reveal changes in their epidemiology as compared to previous studies and highlight the need to set up a warning system to avoid unnoticed PVs.

Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar.

Enterovirus A71 (EV-A71) is a leading cause of hand-foot-and-mouth disease (HFMD) and can be associated with severe neurological complications. EV-A71 strains can be classified into seven genogroups, A-H, on the basis of the VP1 capsid protein gene sequence. Genogroup A includes the prototype strain; genogroups B and C are responsible of major outbreaks worldwide, but little is known about the others, particularly genogroups E and F, which have been recently identified in Africa and Madagascar, respectively. The circulation of EV-A71 in the African region is poorly known and probably underestimated. A rapid and specific assay for detecting all genogroups of EV-A71 is required. In this study, we developed a real-time RT-PCR assay with a competitive internal control (IC). The primers and TaqMan probe specifically target the genomic region encoding the VP1 capsid protein. Diverse EV-A71 RNAs were successfully amplified from the genogroups A, B, C, D, E, and F, with similar sensitivity and robust reproducibility. Neither cross reaction with other EVs nor major interference with the competitive IC was detected. Experimentally spiked stool and plasma specimens provided consistent and reproducible results, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples. The analysis in an African laboratories network of 1889 untyped enterovirus isolates detected 15 EV-A71 of different genogroups. This specific real-time RT-PCR assay provides a robust and sensitive method for the detection of EV-A71 in biological specimens and for the epidemiological monitoring of EV-A71 including its recently discovered genogroups.

Typing of Human Cosaviruses by sequencing of full VP1: Update on global genetic diversity and identification of possible new genotypes circulating in Tunisia, North Africa.

Human Cosaviruses (HCoSVs) are relatively newly characterized picornaviruses; they have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. Previous studies showed HCoSV circulation in Tunisia and only six genotypes circulating in the country were reported. In the present study, we sequenced 27 complete VP1 genomic region from HCoSV isolates in human feces from healthy individuals and patients with acute flaccid paralysis in Tunisia. Most of the Tunisian sequences belong to species A (78%, 21 out of 27). Three sequences belong to species B, two to species E and one sequence to species D. The Tunisian sequences belonged to genotype A6, A7, A8, A10, A1, A17 and E2. Based on genetic distance criteria for assigning genotypes corresponding to neutralization serotypes in enteroviruses we also identified 4 new HCoSV genotypes named A25, B2, B3 and D6. Our study updates the genetic classification of HCoSVs, proposes new genotypes within species A, B and D and contributes to a better knowledge of the HCoSV circulation throughout the world.

Iterative Excretion of Human Cosaviruses from Different Genotypes Associated with Combined Immunodeficiency Disorder.

BACKGROUND: Human cosaviruses (HCoSVs) are newly discovered enteric viruses in the Picornaviridae family. They have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. They remain rarely documented in immunodeficient patients. OBJECTIVES: This study reports iterative excretion of HCoSVs in a patient with major histocompatibility complex (MHC) class II combined immunodeficiency, a relatively common primary immunodeficiency in consanguineous settings. METHODS: A total of 35 samples were collected from a patient followed for oral polio vaccine strains detection in stool samples during a 57-month period. Detection of HCoSVs in stools was performed by nested RT-PCR in the 5' noncoding region. The genotype identification and screening for recombinant strains was performed by sequencing in the VP1 and 3D genomic regions followed by phylogenetic analysis. RESULTS: The patient was infected with HCoSVs twice at a 3-year interval. The excreted viruses belonged to 2 different genotypes with 2 probable recombinant viruses. During HCoSV infections, the patient was also excreting Sabin-related polioviruses. CONCLUSIONS: This study describes excretion kinetics and genetic characteristics of HCoSVs in a patient with combined immunodeficiency due to MHC class II expression defect. The patient did not have concomitant symptoms related to the HCoSV infection.

Molecular epidemiology of coxsackievirus type B1.

Coxsackievirus type B1 (CVB1) has emerged globally as the predominant enterovirus serotype and is associated with epidemics of meningitis and chronic diseases. In this report, the phylogeny of CVB1 was studied based on the VP1 sequences of 11 North African isolates and 81 published sequences. All CVB1 isolates segregated into four distinct genogroups and 10 genotypes. Most of the identified genotypes of circulating CVB1 strains appear to have a strict geographical specificity. The North African strains were of a single genotype and probably evolved distinctly. Using a relaxed molecular clock model and three different population models (constant population, exponential growth and Bayesian skyline demographic models) in coalescent analysis using the BEAST program, the substitution rate in CVB1 varied between 6.95 × 10(-3) and 7.37 × 10(-3) substitutions/site/year in the VP1 region. This study permits better identification of circulating CVB1, which has become one of the most predominant enterovirus serotypes in humans.

Prevalence of human cosaviruses in Tunisia, North Africa.

A new picornavirus, named human Cosavirus (HCoSV) was isolated recently from stools of children with acute flaccid paralysis (AFP) and healthy children in Pakistan and Afghanistan. Since then, it was also isolated from patients from other countries. Five species are presently identified forming a new genus in the Picornaviridae family. This study reports the detection of HCoSV in stool specimens collected as part of the National Poliovirus surveillance Program in Tunisia, between 2011 and 2012, from patients with AFP and healthy individuals among their contacts. One hundred and ninety four stool samples were investigated by RT-PCR in the 5' non-coding region of the genome. A total of 64 specimens (33%) tested positive for HCoSV. HCoSV positive specimens were found in 36 cases with neurological syndromes and 28 of their healthy contacts. The highest rate of HCoSV infection (62.5%) occurred in children younger than 6 years of age. The sampling date of stool specimens suggested that HCoSV infection occurred regularly over time. Also, the sampling origin of stool specimen showed that HCoSV infection was detected in almost all the governorates of Tunisia from the North to the South of the country. This study is the first report of HCoSV prevalence in the North African region. It contributes to a better knowledge on the geographic distribution and the epidemiology of these viruses.

Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5.

Human cosaviruses (HCoSVs) are newly discovered viruses in Picornaviridae family. Until now, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the virus. Nevertheless, no laboratory has yet reported the replication of these viruses in cultured cell lines. In the present work, the propagation of HCoSV strains isolated from human fecal specimens in MRC5 cell line and their induced cytopathic effects (CPE) was studied. The first sign of virus growth was observed 24-48h after inoculation. The cells rounded up and clumped together rapidly; empty areas became visible and, on the third day of CPE, a remarkable decrease in live cells was observed. This represents the first report on in vitro model of HCoSV replication which opens up opportunities for future investigations of these new viruses.

[Genetic variability of type 3 echoviruses]. / Variabilité génétique des échovirus de type 3.

The aim of the present work was to assess the genetic and antigenic variability in the VP1 region of type 3 echviruses (E-3), an enterovirus serotype associated to meningitis, neuro-muscular diseases and type 1 diabetes in human. Forty-six VP1 sequences of E-3, among which 9 were isolated in tunisian infants, were included. Phylogenetic analyses and nucleotidic divergence rates were studied in the complete VP1 region and in a 290-nucleotides fragment in the 5' part of the P1. Aminoacid sequences were deduced in the aim to identify genotype-specific antigenetic determinants. E-3 sequences divided into two genogroups, I and II; the genetic variability within the E-3 serotype reached 29.1%. Genogroup I included sequences with a relatively high genetic diversity among each other, some of them grouped in one genotype with at most 15.1% divergence rate. The sequences included in Genogroup II have a maximum of 13.8% divergence corresponding probably to only one genotype. The two genogroups have a concomitant circulation and a wide geographical and temporal distribution. Aminoacid substitutions that may be specific to genogroups, genotypes and special variant were noted. This work provides a first tentative of classification of E-3 into genogroups and genotypes and reports new E-3 sequences from North Africa. It contributes to a better understanding of the molecular epidemiology of human enteroviruses, and of Echoviruses type 3 in particular, a serotype that remains insufficiently studied in the international literature.

Update on molecular characterization of coxsackievirus B5 strains.

Among Coxsackie B viruses, Coxsckievirus B5 is one of the most predominant serotypes in human, it is frequently associated with cases of neurological diseases, epidemics of meningitis and is a common cause of cardiomyopathy and diabetes. In the present study 27 isolates of Coxsackievirus B5 from North Africa, obtained from cerebrospinal fluid and stool samples of healthy individuals, patients with acute flaccid paralysis or aseptic meningitis were investigated by partial sequencing in the 5' half of the VP1 region and compared to the up-to-date published Coxsackievirus B5 sequences in the same genomic region. Four distinct genomic groups and ten different clusters were individualized. Most of the isolates from Algeria and Tunisia belonged to two clusters. For both, the sequences from North Africa clustered mainly with sequences from European countries, the majority isolated recently during the 2000s. The analysis of the alignment of amino-acids sequences in the VP1 gene revealed four major substitutions in strains from different clusters, we also noticed changes in the BC-loop region; this region is associated with viral antigenicity. This study permit to better identify circulating Coxsackievirus B5 strains throughout the world and their genetic relationship. The protein analysis showed changes that could imply some antigenic significance. J. Med. Virol. 83:1247-1254, 2011. © 2011 Wiley-Liss, Inc.

Phylogenetic analysis of complete VP1 sequences of echoviruses 11 and 6: high genetic diversity and circulation of genotypes with a wide geographical and temporal range.

Echovirus 6 (E6) and echovirus 11 (E11) are common causes of meningitis and other human diseases; they are among the most frequently isolated enteroviruses worldwide. In the present work we have studied genetic variability over the entire VP1 gene of selected isolates representing a wide geographical and temporal range. Fifty new sequences from North Africa were included, together with previously published sequences from different countries. The sequence diversity between strains of the same type was high: 22 and 30 % for E6 and E11, respectively. Phylogenetic analysis revealed five genogroups within each type, the genetic diversity within a genogroup generally being <20 %. Some genogroups were further subdivided into genotypes, most containing isolates that had circulated over a wide geographical (several countries from different continents) and temporal (up to two decades) range. Several genotypes were also shown to co-circulate in a region during the same period of time. These features differ from other enteroviruses that divide into temporal or geographical clusters. This study reports new sequences from North Africa, updates the molecular epidemiology of E6 and E11, and proposes a new genogroup in each type.

Phylogenetic analysis of echovirus 11 in the 3' end of the VP1.

OBJECTIVE: Echovirus 11 is one of the most frequently isolated enterovirus serotypes, causing a wide range of clinical diseases. We studied the genetic diversity in the 3' end of the VP1 gene of strains from different geographical origin in the world. METHODS: The sequences in the 3' end of the VP1 of 11 Tunisian isolates were determined and aligned with the published sequences to establish a phylogenetic profile. RESULTS: The grouping of the sequences was similar to what was previously reported by analyzing the whole VP1 gene with 4 genogroups, designated A-D, and 5 lineages in genogroup D. All Tunisian strains belonged to genogroup D, together with other sequences mainly from the USA and Europe. Contrary to the sequences from the USA isolated during the last 3 decades, which mostly belonged to the D4 lineage, those from Tunisia belonged to different lineages within genogroup D according to their isolation date: isolates from the early 1990s belonged to D3, those of the mid 1990s to D4 and the most recent ones to D5. CONCLUSION: Our findings further widen the interest of partial sequencing in the VP1 to study the molecular epidemiology of echovirus 11 and indicate that the genetic evolution of circulating strains may differ from one country to another according to the region's epidemiological specificities.

Identification of measles virus genotypes from recent outbreaks in countries from the Eastern Mediterranean Region.

BACKGROUND: Molecular characterization of measles viruses (MV) helps to identify transmission pathways of the virus and to document persistence or interruption of endemic virus circulation. In the Eastern Mediterranean Region, measles genotypes from only few countries have been documented. OBJECTIVES: This study reports the genetic characteristics of virus strains from recent measles outbreaks in Tunisia, Libya, Syria and Iran in 2002-2003. STUDY DESIGN: Virus sequences in the nucleoprotein gene were obtained by PCR amplification of virus isolates or serum samples. The sequences were compared to the reference ones for genotype identification and to other published sequences within the same genotype. RESULTS AND CONCLUSIONS: The Tunisian and Libyan epidemic strains belonged to genotype B3, they were closely related to each other and to isolates from Western Africa. The Syrian and Iranian viruses belonged to genotype D4, and differed from each other and from the other published sequences within this genotype. Our results provide valuable baseline and new tools for improved virological measles surveillance in the future, at country, regional and global levels.

Molecular characterization of coxsackievirus B5 isolates.

Coxsackie B viruses of serotype 5 are associated frequently with sporadic cases of neurological diseases, epidemics of meningitis, and chronic diseases such as cardiomyopathy and diabetes. In this article, 15 strains of Coxsackievirus B5 isolated from patients with neurological disorders and healthy people were investigated by partial sequencing in the 5' half of the VP1 region and compared to other published sequences of Coxsackievirus B5, in the same genomic region. All Coxsackievirus B5 sequences showed less than 25% nucleotide difference between each other and a minimum of 27.8% of divergence with prototype sequences from other Coxsackievirus B serotypes. Within the Coxsackievirus B5 group of sequences, four clusters were individualized and may correspond to four genotypes: one genotype with large geographical distribution, containing most recent strains that have circulated from 1984 to 2000, another genotype represented by the prototype Faulkner strain, isolated in the early 1950s, and two intermediate genotypes, comprising strains isolated from 1970 to 1999 and closely related to swine vesicular disease virus. This study confirms the ability of partial sequencing in VP1 to determine serotype and to genetically characterize Coxsackievirus B5 field isolates. It gives a first approach on the molecular epidemiology of these viruses, which have a particular importance in human health.