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The appearance of new variants of SARS-CoV-2 has recently challenged public health authorities with respect to tracking transmission and mitigating the impact in the evolving pandemic across countries. B.1.525 is considered a variant under investigation since it carries specific genetic signatures present in P.1, B.1.1.7, and B.1.351. Here we report genomic evidence of the first likely imported case of the SARS-CoV-2 B.1.525 variant, isolated in a traveler returning from Nigeria., (© 2021 Wiley Periodicals LLC.)

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

The region known as pX in the 3' end of the human T-cell lymphotropic virus type 1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. HTLV-1 ORF-I produces the protein p12 and its cleavage product p8. The functions of these proteins have been linked to immune evasion and viral infectivity and persistence. It is known that the HTLV-1 infection does not necessarily imply the development of pathological processes and here we evaluated whether natural mutations in HTLV-1 ORF-I can influence the proviral load and clinical manifestation of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). For that, we performed molecular characterization, datamining and phylogenetic analysis with HTLV-1 ORF-I sequences from 156 patients with negative or positive diagnosis for HAM/TSP. Our analyses demonstrated that some mutations may be associated with the outcome of HAM/TSP (C39R, L40F, P45L, S69G and R88K) or with proviral load (P34L and F61L). We further examined the presence of mutations in motifs of HBZ and observed that P45L mutation is located within the HBZ nuclear localization signal and was found more frequently between patients with HAM/TSP and high proviral load. These results indicate that some natural mutations are located in functional domains of ORF-I and suggests a potential association between these mutations and the proviral loads and development of HAM/TSP. Therefore it is necessary to conduct functional studies aimed at evaluating the impact of these mutations on the virus persistence and immune evasion., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Human T cell lymphotropic virus type 1 (HTLV-1) infection in Spain is rare and mainly affects immigrants from endemic regions and native Spaniards with a prior history of sexual intercourse with persons from endemic countries. Herein, we report the main clinical and virological features of cases reported in Spain. All individuals with HTLV-1 infection recorded at the national registry since 1989 were examined. Phylogenetic analysis was performed based on the long terminal repeat (LTR) region. A total of 229 HTLV-1 cases had been reported up to December 2012. The mean age was 41 years old and 61% were female. Their country of origin was Latin America in 59%, Africa in 15%, and Spain in 20%. Transmission had occurred following sexual contact in 41%, parenteral exposure in 12%, and vertically in 9%. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) was diagnosed in 27 cases and adult T cell leukemia/lymphoma (ATLL) in 17 subjects. HTLV-1 subtype could be obtained for 45 patients; all but one belonged to the Cosmopolitan subtype a. One Nigerian pregnant woman harbored HTLV-1 subtype b. Within the Cosmopolitan subtype a, two individuals (from Bolivia and Peru, respectively) belonged to the Japanese subgroup B, another two (from Senegal and Mauritania) to the North African subgroup D, and 39 to the Transcontinental subgroup A. Of note, one divergent HTLV-1 strain from an Ethiopian branched off from all five known Cosmopolitan subtype 1a subgroups. Divergent HTLV-1 strains have been introduced and currently circulate in Spain. The relatively large proportion of symptomatic cases (19%) suggests that HTLV-1 infection is underdiagnosed in Spain.

Human T-lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has also been implicated in several disorders, including periodontal disease. The proviral load is an important biological marker for understanding HTLV-1 pathogenesis and elucidating whether or not the virus is related to the clinical manifestation of the disease. This study describes the oral health profile of HTLV-1 carriers and HAM/TSP patients in order to investigate the association between the proviral load in saliva and the severity of the periodontal disease and to examine virus intra-host variations from peripheral blood mononuclear cells and saliva cells. It is a cross-sectional analytical study of 90 individuals carried out from November 2006 to May 2008. Of the patients, 60 were HTLV-1 positive and 30 were negative. Individuals from the HTLV-1 positive and negative groups had similar mean age and social-economic status. Data were analyzed using two available statistical software packages, STATA 8.0 and SPSS 11.0 to conduct frequency analysis. Differences of P < 0.05 were considered statistically significant. HTLV-1 patients had poorer oral health status when compared to seronegative individuals. A weak positive correlation between blood and saliva proviral loads was observed. The mean values of proviral load in blood and saliva in patients with HAM/TSP was greater than those in HTLV-1 carriers. The HTLV-1 molecular analysis from PBMC and saliva specimens suggests that HTLV-1 in saliva is due to lymphocyte infiltration from peripheral blood. A direct relationship between the proviral load in saliva and oral manifestations was observed., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: to describe the genetic diversity of HIV-1 isolates from serum positive women followed up at a reference center., Methods: transversal study, including 96 women with two ELISA serological tests and a Western Blot confirmatory test. The viral charge was determined by the b-DNA kit, and the counting of T CD4 and T CD8 lymphocytes, by the Excalibur flow cytometry, from the samples of peripheral blood. The extraction and purification of pro-viral DNA was performed by the polymerase (PCR) chain reaction, using the QIAamp Blood kit (Qiagen Inc., Chatsworth, CA, U.S.A.). Sequencing of the pol region was done in 52 isolates with the 3100 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA), and the genotyping was assessed by the Rega Subtyping Tool. The resistance pattern to anti-retrovirals (ARV) was inferred by the algorithm from the Stanford HIV Resistance data bank. Participants' clinical stages were defined as A, B or C, according to the criteria established by the Center for Diseases Control (CDC). For statistical analysis, the chi(2) test was used for the categorical variables and the Student's t test, for the numerical variables., Results: The average age of the sample, the disease and treatment average duration were respectively: 33.7 years old, 3.8 and 2.5 years. The viral charge average was log(10) 2.3 copies/mL; the T CD4 e T CD8 lymphocytes, 494.9 cells/microL and 1126.4 cells/microL. Concerning the clinical stage, 30 women were in stage A, 47 in B and 19 in C. Sequencing from the 52 isolates found 33 of B subtype, 4 of F, 1 of C and 14 of BF recombinant. The analysis of resistance to ARV has shown 39 (75.0%) susceptible isolates, 13 (25.0%) resistant to reversal transcriptase inhibitors (RTIN), and 3 (5.7%) resistant to protease inhibitor (PI)., Conclusions: There has been a large variety of HIV-1 and a high percentage of isolates resistant to ARV in the studied sample.

Fas (TNFRSF6/Apo-1/CD95) is a type I transmembrane receptor, which mediates apoptosis. Fas gene mutations, aberrant transcripts, and abundant expression of Fas have been reported in adult T cell leukemia (ATL). To further elucidate the role of Fas in ATL pathogenesis, we investigated whether the -670 FAS promoter A/G polymorphism (STAT1-binding site) might contribute to susceptibility and clinical outcome in ATL. Thirty-one patients with ATL, 33 healthy, human T lymphotropic virus type 1-infected individuals, and 70 healthy, uninfected controls were genotyped for the FAS -670 polymorphism by PCR-restriction fragment-length polymorphism. The AA genotype was significantly over-represented in ATL patients in comparison with healthy controls (P=0.006), as well as asymptomatics (P=0.037), corresponding to an odds ratio (OR) of 3.79 [95% confidence intervals (CI; 1.28-11.41)] and 4.58 [95% CI (1.13-20.03)], respectively. The AA group also comprised significantly more aggressive (acute and lymphoma) clinical subtypes [P=0.012; OR=8.40; 95% CI (1.60-44.12)]. In addition, we observed a statistically significant association between GG genotype and survival (log rank test, P=0.032). Finally, IFN-gamma-induced but not basal FAS mRNA levels were increased significantly (P=0.049) in PBMCs from AA versus GG individuals, demonstrating the IFN-dependent functionality of the -670 polymorphism. In conclusion, our results demonstrate that a functional Fas promoter polymorphism is significantly associated to susceptibility, clinical manifestation, and survival in ATL.

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

The city of Salvador, Bahia, Brazil, has sociodemographic characteristics similar to some African cities. Up to now, it has had the highest prevalence of human T-cell lymphotropic virus type I (HTLV-I) infection (1.74%) in the country. To investigate which strains of HTLV-I are circulating in Salvador, we studied isolates from 82 patients infected with HTLV-I: 19 from the general population, 21 from pregnant women, 16 from intravenous drug users, and 26 from patients and their family attending a neurologic clinic. Phylogenetic analysis from part of the LTR fragments showed that most of these isolates belonged to the Transcontinental subgroup of the Cosmopolitan subtype (HTLV-Ia). Only one sample from a pregnant woman was closely related to the Japanese subgroup, suggesting recent introduction of a Japanese HTLV-I lineage into Salvador. betaA-Globin haplotypes were examined in 34 infected individuals and found to be atypical, confirming the racial heterogeneity of this population. A total of 20 chromosomes were characterized as Central African Republic (CAR) haplotype (29.4%), 31 (45.6%) were characterized as Benin (BEN) haplotype, and 17 (25%) were characterized as Senegal (SEN) haplotype. Five patients' genotypes (14.7%) were CAR/CAR; 10 (29,4%), BEN/BEN; 9 (26.5%), CAR/BEN; 2 (5.9%), BEN/SEN; and 7 (20.6%), SEN/SEN. One patient's genotype (2.9%) was CAR/SEN. The betaA-globin haplotype distribution in Salvador is unusual compared with other Brazilian states. Our data support the hypothesis of multiple post-Columbian introductions of African HTLV-Ia strains in Salvador, Bahia, Brazil.