M2-2 gene as a new alternative molecular marker for phylogenetic, phylodynamic, and evolutionary studies of hRSV.

The human Respiratory Syncytial Virus (hRSV) is the main causative agent of acute respiratory infections (ARI), such as pneumonia and bronchiolitis. One of the factors that lead to success in viral replication is the interaction of the M2-2 protein with the ribosomal complex. This interaction is responsible for the phase change of viral activity, acting as an inhibitor or inducer of viral replication, according to the concentration of mRNA. Based on the importance of M2-2 gene and protein have to viral physiology, we performed here evaluations of genetic diversity, phylogenetic reconstructions, phylodynamics, and selection test. Our results suggested an alternative way of classifying this virus in clades A and B, based on a new phylogenetic marker, the M2-2 gene. Therefore, our study is the first one to investigate the dynamics of the evolutionary diversification process of hRSV from the perspective of the M2-2 viral gene. In our study was also identified that the M2-2 gene is under the effect of purifying selection originated by population genetic bottlenecks. Therefore, the M2-2 gene demonstrated an interesting potential to be applied in evolutionary studies involving hRSV, recovering phylogenetic signals and traits of natural selection under the evolution of this virus.

Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

Genome interaction of the virus and the host genes and non-coding RNAs in SARS-CoV-2 infection.

In this review, we highlight the interaction of SARS-CoV-2 virus and host genomes, reporting the current studies on the sequence analysis of SARS-CoV-2 isolates and host genomes from diverse world populations. The main genetic variants that are present in both the virus and host genomes were particularly focused on the ACE2 and TMPRSS2 genes, and their impact on the patients' susceptibility to the virus infection and severity of the disease. Finally, the interaction of the virus and host non-coding RNAs is described in relation to their regulatory roles in target genes and/or signaling pathways critically associated with SARS-CoV-2 infection. Altogether, these studies provide a significant contribution to the knowledge of SARS-CoV-2 mechanisms of infection and COVID-19 pathogenesis. The described genetic variants and molecular factors involved in host/virus genome interactions have significantly contributed to defining patient risk groups, beyond those based on patients' age and comorbidities, and they are promising candidates to be potentially targeted in treatment strategies for COVID-19 and other viral infectious diseases.

Wa-1 Equine-Like G3P[8] Rotavirus from a Child with Diarrhea in Colombia.

Rotavirus A (RVA) has been considered the main cause of diarrheal disease in children under five years in emergency services in both developed and developing countries. RVA belongs to the Reoviridae family, which comprises 11 segments of double-stranded RNA (dsRNA) as a genomic constellation that encodes for six structural and five to six nonstructural proteins. RVA has been classified in a binary system with Gx[Px] based on the spike protein (VP4) and the major outer capsid glycoprotein (VP7), respectively. The emerging equine-like G3P[8] DS-1-like strains reported worldwide in humans have arisen an important concern. Here, we carry out the complete genome characterization of a previously reported G3P[8] strain in order to recognize the genetic diversity of RVA circulating among infants in Colombia. A near-full genome phylogenetic analysis was done, confirming the presence of the novel equine-like G3P[8] with a Wa-like backbone for the first time in Colombia. This study demonstrated the importance of surveillance of emerging viruses in the Colombian population; furthermore, additional studies must focus on the understanding of the spread and transmission dynamic of this important RVA strain in different areas of the country.

First complete genome sequence and molecular characterization of Canine morbillivirus isolated in Central Brazil.

The Brazilian regions are still highly endemic areas for Canine morbillivirus [canine distemper virus (CDV)]. However, little is known regarding the genetic variability of the strain circulating in several Brazilian regions. Here, we report the first full-length genome and molecular characterization of CDV isolated from domestic dogs in the Brazilian Center-West region. Sequence alignment and phylogenetic analyses based on deduced amino acid and nucleotide sequences showed that the isolated strain is characterized as the South America-I/Europe genotype. However, it segregates into a CDV subgenotype branch. Interestingly, both H and F proteins have a gain of a potential N-glycosylation sites compared to the Onderstepoort vaccine strain. Therefore, this study provides a reference to further understand the epidemic and molecular characteristics of the CDV in Brazil.

Subtyping bovine viral diarrhea virus (BVDV): Which viral gene to choose?

Bovine viral diarrhea virus-1 (BVDV-1, Pestivirus A) and BVDV-2 (Pestivirus B) have been clustered into 21 and 4 subtypes, respectively. This genetic diversity, in addition to the lack of consensus on which genomic region to use for BVDV subtyping, has resulted in conflicting classifications depending on the target analyzed. Here, we investigated which genes or UTRs would reproduce the phylogeny obtained by complete genome (CG) analyses. The study was carried out with 91 (BVDV-1) and 85 (BVDV-2) CG available on GenBank database. The viruses were subtyped by analyzing their CG, as well as their individual genes and UTRs (complete 3' and 5'UTRs, and partial 5'UTR); and the phylogeny results were compared to each other. The sequences were aligned using the ClustalW multiple method (BioEdit Alignment Editor software, v.7.0.5.3) and the phylogenetic analyses were performed by the Maximum Likelihood method (MEGA-X software, v.10.2.4), with 1000 bootstrap replicates. The best analysis model for each gene/UTR was defined using the jModelTest software. The geodesic distance between the CG (reference) and individual genes/UTRs trees was also calculated (TreeCmp software, v.2.0). In general, 3'UTR-based analyses, followed by 5'UTR, presented the least reliable subtyping results. Regarding BVDV-1, phylogeny based on C, Erns, E1, E2, p7, NS2, NS3, NS4B, NS5A and NS5B was consistent with that of CG. In contrast, analyses performed with individual BVDV-2 genes showed at least one different clustering from the phylogeny based on the CG. After analyzing the geodesic distance between the CG and genes/UTRs trees, we observed that NS4B (for BVDV-1) and NS5A (BVDV-2) presented the closest topology and edge length to the CG analyses. Finally, comparing the phylogeny performed with the CG and the genes/UTRs, as well as the geodesic distance between them, we understand that NS4B and NS5A represent the most suitable targets for BVDV-1 and -2 subtyping, respectively, and may be considered in future phylogenetic studies.

Comparison of four extraction methods for the detection of hepatitis B virus DNA in dried blood spot samples.

The dried blood spot (DBS) samples are a useful resource for viral DNA isolation and important in increasing access to HBV diagnosis. However, the choice of the DNA extraction method is crucial for reliable results. We compared the reliability of four DNA extraction methods using DBS samples for the qualitative and quantitative detection of HBV. A panel of serially diluted HBV DNA in whole blood was spotted onto filter paper (Whatman 903 paper and Whatman FTA cards). Four methods were used to extract DNA: QIAamp® DNA Blood Mini Kit (Qiagen); High Pure Viral Nucleic Acid Kit (Roche); Invisorb Spin Blood Midi Kit (Invitek), and DBS Genomic DNA Isolation Kit (Norgen Biotek). Two qualitative PCRs for the core and surface gene regions of HBV were used, and in-house real-time PCR was also evaluated. It was possible to detect HBV DNA using all extraction and PCR protocols. The lowest limit of detection was found using Whatman 903 paper, Roche extraction, and qualitative PCR (20 copies of HBV DNA per ml) for the surface/polymerase HBV region. In the case of in-house real-time PCR, the lowest limit of detection was found using both Roche and Qiagen assays (estimated 2 × 103 copies per ml). These results suggest the importance of both the extraction method and PCR protocol in detecting HBV DNA in DBS. This study provides insights into the utility of DBS samples in HBV molecular diagnosis and their feasibility in low resource areas where cold storage and transportation may be difficult.

Diversity of ß-lactamase-encoding genes in wastewater: bacteriophages as reporters.

A reservoir of antibiotic resistance genes (ARGs) is present in pathogenic, commensal, and environmental bacteria as well as in mobile genetic elements, including bacteriophages. Wastewater treatment plants (WWTPs) are considered hotspots for the spread of ARGs. The aim of this work was to analyze the diversity of the highly prevalent ARGs blaCTX-M and blaTEM in bacterial and bacteriophage fractions associated with human and animal environments through the study of urban waste and animal residues discharged into WWTPs to provide information about the composition and maintenance of the current resistome in Buenos Aires, Argentina. The results showed that a putative extended-spectrum variant of the blaTEM gene was the most frequently detected, with blaTEM-116 being the most prevalent, while a recently described type, blaTEM-229, was also found. In the bacteriophage fraction, we detected blaCTX-M genes from four out of the five clusters described. The detection of blaCTX- M-9-like and blaCTX-M-25-like genes was unexpected based on surveys of the ARGs from clinical pathogens circulating regionally. The finding of divergent blaCTX-M sequences associated with previously reported environmental genes argues in favor of the natural environment as a reservoir of resistance genes. ARGs were detected in bacteriophages as frequently as in bacterial communities, and furthermore, the blaCTX-M genes were more diverse in the bacteriophage fraction. Bacteriophages might therefore play a role in the spread of ARGs in the environment, but they might also be used as "reporters" for monitoring circulating ARGs.

Exploring the rationale for thermotherapy in COVID-19.

Increased transmissibility of the pandemic severe acute respiratory coronavirus 2 (SARS-CoV-2) has been noted to occur at lower ambient temperatures. This is seemingly related to a better replication of most respiratory viruses, including SARS-CoV-2, at lower-than-core body temperatures (i.e., 33 °C vs 37 °C). Also, intrinsic characteristics of SARS-CoV-2 make it a heat-susceptible pathogen. Thermotherapy has successfully been used to combat viral infections in plants which could otherwise result in great economic losses; 90% of viruses causing infections in plants are positive-sense single-stranded ribonucleic acid (+ssRNA) viruses, a characteristic shared by SARS-CoV-2. Thus, it is possible to envision the use of heat-based interventions (thermotherapy or mild-temperature hyperthermia) in patients with COVID-19 for which moderate cycles (every 8-12 h) of mild-temperature hyperthermia (1-2 h) have been proposed. However, there are potential safety and mechanistic concerns which could limit the use of thermotherapy only to patients with mild-to-moderate COVID-19 to prevent disease progression rather than to treat patients who have already progressed to severe-to-critical COVID-19. Here, we review the characteristics of SARS-CoV-2 which make it a heat-susceptible virus, potential host mechanisms which could be enhanced at higher temperatures to aid viral clearance, and how thermotherapy could be investigated as a modality of treatment in patients with COVID-19 while taking into consideration potential risks.

Molecular features similarities between SARS-CoV-2, SARS, MERS and key human genes could favour the viral infections and trigger collateral effects.

In December 2019, rising pneumonia cases caused by a novel ß-coronavirus (SARS-CoV-2) occurred in Wuhan, China, which has rapidly spread worldwide, causing thousands of deaths. The WHO declared the SARS-CoV-2 outbreak as a public health emergency of international concern, since then several scientists are dedicated to its study. It has been observed that many human viruses have codon usage biases that match highly expressed proteins in the tissues they infect and depend on the host cell machinery for the replication and co-evolution. In this work, we analysed 91 molecular features and codon usage patterns for 339 viral genes and 463 human genes that consisted of 677,873 codon positions. Hereby, we selected the highly expressed genes from human lung tissue to perform computational studies that permit to compare their molecular features with those of SARS, SARS-CoV-2 and MERS genes. The integrated analysis of all the features revealed that certain viral genes and overexpressed human genes have similar codon usage patterns. The main pattern was the A/T bias that together with other features could propitiate the viral infection, enhanced by a host dependant specialization of the translation machinery of only some of the overexpressed genes. The envelope protein E, the membrane glycoprotein M and ORF7 could be further benefited. This could be the key for a facilitated translation and viral replication conducting to different comorbidities depending on the genetic variability of population due to the host translation machinery. This is the first codon usage approach that reveals which human genes could be potentially deregulated due to the codon usage similarities between the host and the viral genes when the virus is already inside the human cells of the lung tissues. Our work leaded to the identification of additional highly expressed human genes which are not the usual suspects but might play a role in the viral infection and settle the basis for further research in the field of human genetics associated with new viral infections. To identify the genes that could be deregulated under a viral infection is important to predict the collateral effects and determine which individuals would be more susceptible based on their genetic features and comorbidities associated.

High molecular prevalence of HPV and other sexually transmitted infections in a population of asymptomatic women who work or study at a Brazilian university.

Sexually transmitted infections (STIs) represent a global health problem with variable prevalence depending on the geographical region and the type of population. Human papillomavirus (HPV) encompasses widespread virus types related to cervical carcinogenesis. The present study investigated the molecular prevalence of HPV and seven other important STIs in asymptomatic women working or studying at a Brazilian university. A secondary aim was to assess cytological abnormalities associated with HPV and other STIs coinfections. We recruited 210 women from a Brazilian university. HPV was detected using a single-round polymerase chain reaction (sPCR) followed by a viral genotyping by restriction fragment length polymorphism (RFLP-PCR). The presence of seven STIs: Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1 and HSV-2 was detected by multiplex PCR (M-PCR). Furthermore, cytological findings and epidemiological characteristics were evaluated.The mean age of the participants was 27.1 years old. HPV prevalence was 33.8%, and HPV16 was the most frequently detected papillomavirus genotype. Moreover, multiple HPV infections were common (42.2%). We detected at least one STI agent in 11.4% of the tested women, most frequently C. trachomatis (6.7%). Among HPV-positive women, 14.1% were coinfected with other STI agents. Cytological abnormalities were observed in 9.5% of smears, and HPV-DNA, high-risk HPV (HR-HPV), HPV16 and HPV multiple infections were associated with abnormal cytological findings. There was a high prevalence of HPV, and C. trachomatis was the most prevalent STI agent, with low rates of cytological abnormalities. These findings highlight the need of timely STI diagnosis in young asymptomatic women and of a public policy design for STI prevention.

Genomic analyses of Biston suppressaria nucleopolyhedrovirus: a viral isolate obtained from the tea looper caterpillar, Biston suppressaria (Guenée, 1857).

We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology.

Effective methylation triage of HPV positive women with abnormal cytology in a middle-income country.

The S5-methylation test, an alternative to cytology and HPV16/18 genotyping to triage high-risk HPV-positive (hrHPV+) women, has not been widely validated in low-middle-income countries (LMICs). We compared S5 to HPV16/18 and cytology to detect cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) and CIN3+ in hrHPV+ women selected from a randomized pragmatic trial of 2661 Colombian women with an earlier-borderline abnormal cytology. We included all hrHPV+ CIN2 and CIN3+ cases (n = 183) age matched to 183

A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit.

The World Health Organization (WHO) has declared a pandemic caused by a new coronavirus named SARS-CoV-2. The growing demand for commercial kits used for automated extraction of SARS-CoV-2 RNA, a key step before rRT-PCR diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples. Although the recommendation is to use automated methods for nucleic acid extraction, alternatives are necessary to replace commercial kits. However, these alternatives should be as reliable as automated methods. This work describes a simple method to detect SARS-CoV-2 from specimens collected in different preservation media. Samples were previously inactivated by heating and precipitating with a PEG/NaCl solution before rRT-PCR assays for Orf1ab, N and S genes. The new method was compared with an automated protocol of nucleic acid extraction. Both procedures showed similar analytical results. Consequently, this simple and inexpensive method is a suitable procedure for laboratory diagnosis of SARS-CoV-2 infection.

Insights on Zika virus envelope gene conservation in American outbreaks.

Phylogenetic studies with Zika virus (ZIKV) have been conducted in Brazil. In this study, we sequenced 8 new sequences of the ZIKV envelope (E) gene from strains of cases from the Paraná and Mato Grosso do Sul states in 2016. A low phylogenetic signal was observed, with more than 40% of unresolved quartets, and the Maximum Likelihood Tree grouped all sequences in the Brazilian branches within the Asian genotype. In addition, a Shannon entropy analysis was conducted, showing a high stability in the E protein through the ZIKV polyprotein. Taken together, these results suggest a high degree of conservation in the ZIKV E gene from the recent American outbreaks.

Betacoronavirus Genomes: How Genomic Information has been Used to Deal with Past Outbreaks and the COVID-19 Pandemic.

In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of coronaviruses in the aftermath of the 2002-2003 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-2020 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2. We focus on shared genomic features of the betacoronaviruses and the application of genomic information to phylogenetic analysis, molecular epidemiology and the design of diagnostic systems, potential drugs and vaccine candidates.

The Concerted Action of Two B3-Like Prophage Genes Excludes Superinfecting Bacteriophages by Blocking DNA Entry into Pseudomonas aeruginosa.

In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host's cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.

Unexpected rabies variant identified in kinkajou (Potos flavus), Mato Grosso, Brazil.

A second case of a novel rabies variant described once in a capuchin monkey from Mato Grosso, Brazil, was discovered in a rabid wild kinkajou from the same region, indicating a public health risk following exposure to either of the two animals.

Zika structural genes determine the virulence of African and Asian lineages.

The Asian lineage of Zika virus (ZIKV) is responsible for the recent epidemics in the Americas and severe disease, whereas the African lineage of ZIKV has not been reported to cause epidemics or severe disease. We constructed a cDNA infectious clone (IC) of an African ZIKV strain, which, together with our previously developed Asian ZIKV strain IC, allowed us to engineer chimeric viruses by swapping the structural and non-structural genes between the two lineages. Recombinant parental and chimeric viruses were analyzed in A129 and newborn CD1 mouse models. In the A129 mice, the African strain developed higher viremia, organ viral loading, and mortality rate. In CD1 mice, the African strain exhibited a higher neurovirulence than the Asian strain. A chimeric virus containing the structural genes from the African strain is more virulent than the Asian strain, whereas a chimeric virus containing the non-structural genes from the African strain exhibited a virulence comparable to the Asian strain. These results suggest that (i) African strain is more virulent than Asian strain and (ii) viral structural genes primarily determine the virulence difference between the two lineages in mouse models. Other factors may contribute to the discrepancy between the mouse and epidemic results.

Soil microbial diversity drops with land-use change in a high mountain temperate forest: a metagenomics survey.

Land-use change has been identified as the most severe threat to biodiversity. Soils are important biodiversity reservoirs, but to what extent conversion of high-altitude temperate forest to arable land affects taxonomic and functional soil biodiversity is still largely unknown. Shotgun metagenomics was used to determine the taxonomic and functional diversity of bacteria, archaea and DNA virus in terms of effective number of species in high-altitude temperate oak and pine-oak forest and arable soils from Mexico. Generally, the soil ecosystem maintained its microbial species richness notwithstanding land-use change. Archaea diversity was not affected by land-use change, but the bacterial diversity decreased with 45-55% when the oak forest was converted to arable land and 65-75% when the pine-oak forest was. Loss in bacterial diversity as a result of land-use change was positively correlated (R2 = 0.41) with the 10-25% loss in functional diversity. The archaeal communities were evener than the bacterial ones, which might explain their different response to land-use change. We expected a decrease in DNA viral communities as the bacterial diversity decreased, i.e. their potential hosts. However, a higher viral diversity was found in the arable than in the forest soils. It was found that converting high altitude oak and pine-oak forests to arable land more than halved the bacterial diversity, but did not affect the archaeal and even increased the viral diversity.