Progressive Vaccinia Acquired through Zoonotic Transmission in a Patient with HIV/AIDS, Colombia.

In March 2015, a patient in Colombia with HIV/AIDS was hospitalized for disseminated ulcers after milking cows that had vesicular lesions on their udders. Vaccinia virus was detected, and the case met criteria for progressive vaccinia acquired by zoonotic transmission. Adherence to an optimized antiretroviral regimen resulted in recovery.

Genomic Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2, Colombia.

Coronavirus disease (COVID-19) in Colombia was first diagnosed in a traveler arriving from Italy on February 26, 2020. However, limited data are available on the origins and number of introductions of COVID-19 into the country. We sequenced the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from 43 clinical samples we collected, along with another 79 genome sequences available from Colombia. We investigated the emergence and importation routes for SARS-CoV-2 into Colombia by using epidemiologic, historical air travel, and phylogenetic observations. Our study provides evidence of multiple introductions, mostly from Europe, and documents >12 lineages. Phylogenetic findings validate the lineage diversity, support multiple importation events, and demonstrate the evolutionary relationship of epidemiologically linked transmission chains. Our results reconstruct the early evolutionary history of SARS-CoV-2 in Colombia and highlight the advantages of genome sequencing to complement COVID-19 outbreak investigations.

Molecular characterization of dengue virus reveals regional diversification of serotype 2 in Colombia.

Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013-2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification.

Detection and Molecular Characterization of Zoonotic Poxviruses Circulating in the Amazon Region of Colombia, 2014.

During 2014, cutaneous lesions were reported in dairy cattle and farmworkers in the Amazon Region of western Colombia. Samples from 6 patients were analyzed by serologic and PCR testing, and results demonstrated the presence of vaccinia virus and pseudocowpox virus. These findings highlight the need for increased poxvirus surveillance in Colombia.

Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform.

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription.

Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs.

Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs.

Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction.

INTRODUCTION: Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. OBJECTIVE: To develop and test a control RNA for YFV detection through real-time RT-PCR. METHODS: A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. RESULTS: A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). CONCLUSION: The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.

Novel Putative Tymoviridae-like Virus Isolated from Culex Mosquitoes in Colombia.

The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5'/3' RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3' end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

Phylogenetic reconstruction of dengue virus type 2 in Colombia.

BACKGROUND: Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and sub-tropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2) has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E) gene from 48 Colombian isolates. RESULTS: As expected, the oldest isolates belonged to the American genotype (subtype V), but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb) as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. CONCLUSION: After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated.

Molecular characterization of mosquitoes (Diptera: Culicidae) from the Colombian rainforest.

A few studies have carried out the taxonomic and molecular characterization of sylvatic mosquito species in Latin America, where some species have been incriminated as vectors for arboviruses and parasites transmission. The present study reports the molecular characterization of mosquito species in the Sierra Nevada de Santa Marta, a natural ecosystem in the Northern coast of Colombia. Manual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. The COI marker was used for species confirmation, and phylogenetic analysis was performed using the neighbor-joining method, with the Kimura-2-Parameters model. Aedes serratus , Psorophora ferox , Johnbelkinia ulopus , Sabethes chloropterus , Sabethes cyaneus , Wyeomyia aporonoma , Wyeomyia pseudopecten , Wyeomyia ulocoma and Wyeomyia luteoventralis were identified. We assessed the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed the identification at the species level. Classical and molecular taxonomy demonstrated to be useful and complementary when morphological characteristics are not well preserved, or the taxonomic group is not represented in public molecular databases.

Novel Highly Divergent SARS-CoV-2 Lineage With the Spike Substitutions L249S and E484K.

COVID-19 pandemics has led to genetic diversification of SARS-CoV-2 and the appearance of variants with potential impact in transmissibility and viral escape from acquired immunity. We report a new and highly divergent lineage containing 21 distinctive mutations (10 non-synonymous, eight synonymous, and three substitutions in non-coding regions). The amino acid changes L249S and E484K located at the CTD and RBD of the Spike protein could be of special interest due to their potential biological role in the virus-host relationship. Further studies are required for monitoring the epidemiologic impact of this new lineage.

Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity has the potential to impact the virus transmissibility and the escape from natural infection- or vaccine-elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in Colombia between January and April 2021, were processed for genome sequencing and lineage determination following the nanopore amplicon ARTIC network protocol and PANGOLIN pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a variant of interest (VOI) with the accumulation of several substitutions affecting the Spike protein, including the amino acid changes I95I, Y144T, Y145S and the insertion 146 N in the N-terminal domain, R346K, E484K and N501Y in the Receptor binding Domain (RBD) and P681H in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and fixation in a relatively short time in Magdalena, Atlantico, Bolivar, Bogotá D.C, and Santander that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies will be required to assess the biological and epidemiologic roles of the substitution pattern found in the B.1.621 lineage.

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia.

BACKGROUND: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70's when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. RESULTS: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major amino acid changes in the analyzed region were found. CONCLUSION: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.

Efficient Method for Molecular Characterization of the 5' and 3' Ends of the Dengue Virus Genome.

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5' and 3' ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5' and 3' ends of dengue virus types 1 to 4. The 5' and 3' ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5' end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3' end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5' and 3' ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.

Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in South America.

SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemic. The virus crossed the species barrier and established in the human population taking advantage of the spike protein high affinity for the ACE receptor to infect the lower respiratory tract. The Nucleocapsid (N) and Spike (S) are highly immunogenic structural proteins and most commercial COVID-19 diagnostic assays target these proteins. In an unpredictable epidemic, it is essential to know about their genetic variability. The objective of this study was to describe the substitution frequency of the S and N proteins of SARS-CoV-2 in South America. A total of 504 amino acid and nucleotide sequences of the S and N proteins of SARS-CoV-2 from seven South American countries (Argentina, Brazil, Chile, Ecuador, Peru, Uruguay, and Colombia), reported as of June 3, and corresponding to samples collected between March and April 2020, were compared through substitution matrices using the Muscle algorithm. Forty-three sequences from 13 Colombian departments were obtained in this study using the Oxford Nanopore and Illumina MiSeq technologies, following the amplicon-based ARTIC network protocol. The substitutions D614G in S and R203K/G204R in N were the most frequent in South America, observed in 83% and 34% of the sequences respectively. Strikingly, genomes with the conserved position D614 were almost completely replaced by genomes with the G614 substitution between March to April 2020. A similar replacement pattern was observed with R203K/G204R although more marked in Chile, Argentina and Brazil, suggesting similar introduction history and/or control strategies of SARS-CoV-2 in these countries. It is necessary to continue with the genomic surveillance of S and N proteins during the SARS-CoV-2 pandemic as this information can be useful for developing vaccines, therapeutics and diagnostic tests.

Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia.

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3' end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3' end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Novel pan-serotype control RNA for dengue virus typing through real-time reverse transcription-polymerase chain reaction.

Dengue virus (DENV) is the causative agent of one of the most important febrile illnesses worldwide. Four DENV serotypes are responsible for a broad clinical spectrum of the disease. Positive controls are costly and required for the validation of molecular test results of DENV serotyping. In this study, we describe the in silico design of the qDENV-Control plasmid with the target sequences to oligonucleotides and probes widely used for DENV serotyping, and the subsequent production of qDENV Control RNA by T7-driven run-off in vitro transcription. The qDENV Control RNA was successfully used to validate the positive and negative DENV serotyping results, allowing its incorporation in routine in-house protocols for virologic surveillance. This Control RNA allowed the absolute quantification of viral RNA copies from unknown samples as required in several fundamental studies.

Complete Genome Sequence of a Colombian Zika Virus Strain Obtained from BALB/c Mouse Brain after Intraperitoneal Inoculation.

A Zika virus (ZIKV) strain was isolated from an acute febrile patient during the Zika epidemics in Colombia. The strain was intraperitoneally inoculated into BALB/c mice, and 7 days postinoculation, neurological manifestations and ZIKV infection in the brain were demonstrated. The reported genome sequence is highly related to strains circulating in the Americas.

Simultaneous circulation of genotypes I and III of dengue virus 3 in Colombia.

BACKGROUND: Dengue is a major health problem in tropical and subtropical regions. In Colombia, dengue viruses (DENV) cause about 50,000 cases annually, 10% of which involve Dengue Haemorrhagic Fever/Dengue Shock Syndrome. The picture is similar in other surrounding countries in the Americas, with recent outbreaks of severe disease, mostly associated with DENV serotype 3, strains of the Indian genotype, introduced into the Americas in 1994. RESULTS: The analysis of the 3'end (224 bp) of the envelope gene from 32 DENV-3 strains recently recovered in Colombia confirms the circulation of the Indian genotype, and surprisingly the co-circulation of an Asian-Pacific genotype only recently described in the Americas. CONCLUSION: These results have important implications for epidemiology and surveillance of DENV infection in Central and South America. Molecular surveillance of the DENV genotypes infecting humans could be a very valuable tool for controlling/mitigating the impact of the DENV infection.

[Zoonotic vaccinia in Colombia: Cumulative evidence of the emergence of poxviruses in the world]. / Vaccinia zoonótica en Colombia: evidencia acumulativa de la emergencia de los poxvirus en el mundo.

The recent occurrence of vaccinia virus infections in humans and animals in Colombia, together with that reported for this and other species of the genus Orthopoxvirus in some South American, African, Asian and European countries, is supporting evidence of the emergence and re-emergence of the genus. This fact has become of great interest for public health around the world due to its biological and an epidemiological features, as was in the past the variola virus, one of its representatives. The emergence and re-emergence of the genus Orthopoxvirus may be a consequence of stopping vaccination against the variola virus in the 1970s and 1980s. This vaccination unsuspectedly induced cross-protective immunity to other species of that genus. This is a review of the history, biology and epidemiology of the main species of the genus Orthopoxvirus, together with its clinical presentation, social context and public health impact in the past, present and future.

La reciente ocurrencia de infecciones por el virus vaccinia en animales y humanos en distintos lugares de la geografía colombiana, sumadas a otras por éste y por otros virus pertenecientes al género Orthopoxvirus (familia Poxviridae), ocurridas en algunos países de Suramérica, África, Asia y Europa se convierten en evidencia de la inminente emergencia y re-emergencia de este género, con características biológicas y epidemiológicas que le confieren gran interés para la salud pública del mundo, como lo fue en el pasado una de sus especies representativas: el virus de la viruela. Esta emergencia y re-emergencia parecen estar relacionadas con la suspensión en las décadas de los 70s y 80s de las campañas de vacunación contra la viruela, las cuales; insospechadamente estuvieron protegiendo a la población, no únicamente contra este virus, sino contra otros del mismo género. En el presente artículo se hace una revisión de la biología y epidemiología de los principales miembros del género Orthopoxvirus, su presentación clínica, antecedentes históricos, contexto social, e impacto en la salud pública mundial en el pasado, presente y a futuro.