Relative frequency of genomic mutations in SARS-CoV-2 recovered from southern Brazilian cases of COVID-19 through the Gamma, Delta and Omicron waves.

The presence of different mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can be related to changes in coronavirus disease (COVID-19) infection. Besides, these viral alterations associated with factors such as massive number of positive cases, vaccination and reinfections can be important in the viral evolution process. As well as, mutations found at low frequencies may have a more neutral action and consequently be less inclined to negative selection, facilitating their spread through the population. Related to that, we aimed to present mutations that are possibly relevant in the process of viral evolution found in 115 SARS-CoV-2 sequences from samples of individuals residing in the metropolitan region of Porto Alegre in the state of Rio Grande do Sul, Brazil. The genome from clinical samples was sequenced using High-Throughput Sequencing (HTS) and analyzed using a workflow to map reads and find variations/SNPs. The samples were separated into 3 groups considering the sample lineage. Of the total number of analyzed sequences, 35 were from the Gamma lineage, 35 from Delta and 45 from Omicron. Amino acid changes present in frequencies lower than 80% of the reads in the sequences were evaluated. 11 common mutations among the samples were found in the Gamma lineage, 1 in the ORF1ab gene, 7 in the S gene, 2 in the ORF6 gene and 1 in the ORF7a gene. While in the Delta lineage, a total of 11 mutations distributed in the ORF1ab, S, ORF7a and N genes, 2, 7, 1 and 1 mutation were found in each gene, respectively. And finally, in the Omicron, 16 mutations were identified, 2 in the ORF1ab gene, 12 in the S gene and 2 in the M gene. In conclusion, we emphasize that genomic surveillance can be a useful tool to assess how mutations play a key role in virus adaptation, and its process of susceptibility to new hosts showing the possible signs of viral evolution.

Evaluation of Mastadenovirus and Rotavirus Presence in Phyllostomid, Vespertilionid, and Molossid Bats Captured in Rio Grande do Sul, Southern Brazil.

Bat-borne viruses may affect public health and the global economy. These mammals have a wide geographical distribution and unique biological, physiological, and immunogenic characteristics, allowing the dissemination of many known and unknown viruses. Enteric viruses, such as adeno (AdV) and rotaviruses, are recognized as the main causative agents of disease and outbreaks. In the present study, the presence of viruses from Adenoviridae and Reoviridae families was evaluated in molossid, phyllostomid, and vespertilionid bats captured in Rio Grande do Sul, Southern Brazil, between September 2021 and July 2022. Sixty bat rectal swabs were analyzed by PCR. Eight (13.3%) samples were positive for adenovirus and classified as human mastadenovirus C (HAdV-C) (three samples) and HAdV-E (five samples) by sequencing followed by phylogenetic analysis. All samples were negative in rotavirus specific RT-PCR. This is the first study to describe the presence of HAdV in samples of Glossophaga soricina, Eptesicus brasiliensis, and Histiotus velatus. Furthermore, the presence of HAdV-E in bats was reported, which is unusual and may suggest that other HAdV genotypes, in addition to HAdV-C, may also be harbored by wild animals. The data generated in the present study reinforces the importance of eco-surveillance of viral agents related to diseases in humans and wild animals. In addition, it is essential to identify possible new hosts or reservoirs that increase the risk of spillover and dissemination of infectious pathogens, helping to prevent and control zoonotic diseases.

Diversity of Omicron sublineages and clinical characteristics in hospitalized patients in the southernmost state of Brazil.

BACKGROUND: Omicron has become the dominant variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since first reported in November 2021. From the initially detected Wuhan lineage, sublineages BA.2, BA.4, BA.5, BQ, XAG, and XBB have emerged over time and are dominant in many countries. Therefore, the aim is to evaluate which variants are circulating and the clinical characteristics of inpatients infected with the Omicron variant. METHODS: This retrospective cohort study selected hospitalized patients admitted with respiratory symptoms to a hospital in the state of Rio Grande do Sul, Brazil, between June and July 2022. SARS-CoV-2 results were analyzed together with clinical outcomes and vaccination status. A viral genome library was prepared and forwarded to the Illumina MiSeq Platform for sequencing. RESULTS: In total, 37 genomes were sequenced. Concerning the Omicron sublineages, our study detected: BA.1 (21 K), BA.2 (21 L), BA.4 (22A), BA.5 (22B), BA.2.12.1 (22C), BQ.1 (22E), XBB (22F), and XAG recombinant. Omicron BA.5 (30%), BA.2 (19%), and BQ.1 (19%) were the most frequent sublineages, respectively. In total, 38% of patients present hypertension, and the most common symptoms were coughing (62%). Analyzing the COVID-19 vaccination, 30% of patients were fully vaccinated, 49% had a partial vaccination status, and 21% were unvaccinated (no dose). CONCLUSIONS: BA.5 was the most prevalent sublineage in our study and surpassed the predominance of BA.2, as reported by the national genomic surveillance program. BQ.1 was diagnosed earlier in this study than it was officially reported in the state. Current data have demonstrated that the Omicron variant causes less severe infections, with the high rate of transmissibility and mutational landscape causing the rapid emergence of new sublineages.

DENV-1 genotype V linked to the 2022 dengue epidemic in Southern Brazil.

Even though Brazil is a country where the dengue virus (DENV) is endemic, until recently, Southern states did not have significant viral circulation, such as Rio Grande do Sul (RS), and some municipalities were even considered dengue-free. During 2022, these places have shown a sharp increase in the incidence of the disease, apparently following a worldwide growth pattern. Therefore, in this study, we monitor and characterize the genetic diversity of DENV circulating in southern Brazil through next-generation sequencing during an outbreak in 2022. We generated 70 DENV-1 genome sequences, all characterized as genotype V, divided into two clade clusters in the L1 lineage. Furthermore, unique mutations have been described in each clade of L1 lineage. Our results are essential in managing outbreaks since these data provide important information during the emergence of DENV circulation in RS. Since the south of Brazil has a lower viral circulation when compared to other Brazilian states, RS still lacks data that can help in understanding the transmission, dissemination, and evolution of the dengue virus. Hence, genomic surveillance efforts are essential to increase the accuracy of preventive actions and to control viral dissemination.

Chikungunya virus infection in the southernmost state of Brazil was characterised by self-limited transmission (2017-2019) and a larger 2021 outbreak.

BACKGROUND: Chikungunya is a mosquito-borne virus that has been causing large outbreaks in the Americas since 2014. In Brazil, Asian-Caribbean (AC) and East-Central-South-African (ECSA) genotypes have been detected and lead to large outbreaks in several Brazilian states. In Rio Grande do Sul (RS), the southernmost state of Brazil, the first cases were reported in 2016. OBJECTIVES AND METHODS: We employed genome sequencing and epidemiological investigation to characterise the Chikungunya fever (CHIKF) burden in RS between 2017-2021. FINDINGS: We detected an increasing CHIKF burden linked to travel associated introductions and communitary transmission of distinct lineages of the ECSA genotype during this period. MAIN CONCLUSIONS: Until 2020, CHIKV introductions were most travel associated and transmission was limited. Then, in 2021, the largest outbreak occurred in the state associated with the introduction of a new ECSA lineage. CHIKV outbreaks are likely to occur in the near future due to abundant competent vectors and a susceptible population, exposing more than 11 million inhabitants to an increasing infection risk.

Reinfection by SARS-CoV-2 by divergent Omicron sublineages, 16 days apart.

Since the beginning of the SARS-CoV-2 pandemic, studies on the variants and sublineages stand out, mainly in the cases of reinfection in a short period. In this study, we describe a case of infection by BA.1.1 sublineage in an individual from Southern Brazil. The same patient acquired reinfection with sublineage BA.2 within 16 days after the first detection. The viral extraction and RT-qPCR were performed on the samples LMM72045 (collected in May 2022) and LMM72044 (collected in June 2022). After the confirmation of SARS-CoV-2 infection, we conducted the sequencing and viral genome analysis. This case of reinfection affected a 52-year-old male patient, without comorbidities, with three doses of vaccines against COVID-19, showing symptoms on May 19. These symptoms lasted for approximately six days. The patient returned to work activities on May 30. However, on June 4, the patient felt a new round of clinical signs that lasted for approximately seven days. Analysis of the viral genomes recovered from patients' clinical samples revealed that the two COVID-19 episodes were related to two divergent VOC Omicron sublineages, namely, BA.1.1 for the first round of symptoms and BA.2 for the second infection. Based on our findings, we can say that the present case of reinfection is the shortest described so far.

Genome of a husavirus from Southern Brazil.

New viruses of the Picornavirales order have been discovered with the increase in the number of sequences obtained by high-throughput sequencing, as well as human stool-associated RNA virus (husavirus [HuV]), found in human stool samples. However, there is much to be clarified about HuV. Its cellular host, evolutionary history, and other biological characteristics are still unknown. Therefore, samples collected from human beings and environmental samples in a watershed in Southern Brazil were processed for the metagenomic library. Upon metagenomic analysis, we identified a HuV (husavirus LMM_67754 OP019707) genome with 8,846 bp, which was reported for the first time in Southern Brazil. The new genome presents only 37% of nucleotide identity with Brazilian strains and more than 90% with genomes from China, Vietnam, Venezuela, and the Netherlands. The HuV phylogeny presents significant differences among genomes, probably because multiple introductions of the virus may have occurred. Many questions still need to be answered about HuV. Therefore, more sequences and studies on this virus are necessary to improve the comprehension of the unknown origin of Picornavirales.

Dynamics of nosocomial SARS-CoV-2 transmissions: Facing the challenge of variants of concern in a Brazilian reference hospital.

The hospital environment can be considered a high risk for the occurrence of SARS-CoV-2 transmission outbreaks, either for health professionals who are directly involved in the care of suspected or confirmed cases of the disease, or for patients, for being in an environment more vulnerable to the acquisition of nosocomial infections. In this molecular epidemiology study, we aimed to analyze the occurrence and transmission dynamics of SARS-CoV-2 in outbreaks and local chains of transmission in a large tertiary teaching hospital in southern Brazil, in addition to verifying circulating strains and their epidemiological relation in the local context, from September 21, 2020 to October 5, 2021. Positive samples involved in COVID-19 clusters or outbreaks were analyzed using clinical, epidemiological and genomic data. Different lineages and sublineages among patients in the same room were observed. Most patients had their first clinical manifestation, evidence of suspicion, and diagnostic confirmation within 7-14 days or >14 days after hospital admission. The patients who have contact with confirmed cases of COVID-19 spent, on average, 6.28 days in the same environment until the positive test. There was a significant association between the outcome and the number of vaccine doses (p < 0.05), where those who received two doses presented a lower occurrence of death. There was a total replacement of variant of concern (VOC) Gamma by VOC Delta from August 2021 at the study site. Although the epidemiological analysis indicates nosocomial infections, through genomic sequencing, it was established that most of the hospital outbreaks had different origins. These findings highlight the utility of integrating epidemiological and genomic data to identify possible routes of viral entry and dissemination.

Chikungunya virus infection in the southernmost state of Brazil was characterised by self-limited transmission (2017-2019) and a larger 2021 outbreak

BACKGROUND Chikungunya is a mosquito-borne virus that has been causing large outbreaks in the Americas since 2014. In Brazil, Asian-Caribbean (AC) and East-Central-South-African (ECSA) genotypes have been detected and lead to large outbreaks in several Brazilian states. In Rio Grande do Sul (RS), the southernmost state of Brazil, the first cases were reported in 2016. OBJECTIVES AND METHODS We employed genome sequencing and epidemiological investigation to characterise the Chikungunya fever (CHIKF) burden in RS between 2017-2021. FINDINGS We detected an increasing CHIKF burden linked to travel associated introductions and communitary transmission of distinct lineages of the ECSA genotype during this period. MAIN CONCLUSIONS Until 2020, CHIKV introductions were most travel associated and transmission was limited. Then, in 2021, the largest outbreak occurred in the state associated with the introduction of a new ECSA lineage. CHIKV outbreaks are likely to occur in the near future due to abundant competent vectors and a susceptible population, exposing more than 11 million inhabitants to an increasing infection risk.

Genome of a husavirus from Southern Brazil

ABSTRACT New viruses of the Picornavirales order have been discovered with the increase in the number of sequences obtained by high-throughput sequencing, as well as human stool-associated RNA virus (husavirus [HuV]), found in human stool samples. However, there is much to be clarified about HuV. Its cellular host, evolutionary history, and other biological characteristics are still unknown. Therefore, samples collected from human beings and environmental samples in a watershed in Southern Brazil were processed for the metagenomic library. Upon metagenomic analysis, we identified a HuV (husavirus LMM_67754 OP019707) genome with 8,846 bp, which was reported for the first time in Southern Brazil. The new genome presents only 37% of nucleotide identity with Brazilian strains and more than 90% with genomes from China, Vietnam, Venezuela, and the Netherlands. The HuV phylogeny presents significant differences among genomes, probably because multiple introductions of the virus may have occurred. Many questions still need to be answered about HuV. Therefore, more sequences and studies on this virus are necessary to improve the comprehension of the unknown origin of Picornavirales.

Genomic and epidemiologic surveillance of SARS-CoV-2 in Southern Brazil and identification of a new Omicron-L452R sublineage.

Recently, SARS-CoV-2 Omicron variant (B.1.1.529) was first identified in Botswana in November 2021. In a short period of time, this highly mutated variant replaced the previous dominant Delta variant, causing an exponential increase in the number of COVID-19 cases, resulting in a new wave of pandemic. This current research article aims to analyze and summarize information about the genetic characteristics, amino acid mutations and epidemiological data providing scientific findings to enrich the SARS-CoV-2 knowledge. More importantly, we describe here, for the first time, the identification of a new Omicron variant of concern: Omicron-L452R in Brazil.

Viral isolation allows characterization of early samples of SARS-CoV-2 lineage B1.1.33 with unique mutations (S: H655Y and T63N) circulating in Southern Brazil in 2020.

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.

Brief dispersion of a putative B.1.1.28-derived SARS-CoV-2 lineage harboring additional N234P and E471Q spike protein mutations in individuals crossing the Argentina-Brazil border.

BACKGROUND: SARS-CoV-2, the virus that causes COVID-19, is constantly mutating, leading to new variants that culminate in a temporal lineages fluctuation. B.1.1.28 lineage has been evolving in Brazil since February 2020 and originated P.1 (VOC), P.2 (VOI) and other P.Xs proposed as new variants. METHODS AND RESULTS: In this study, through the Illumina platform, we performed the whole-genome sequencing of 26 positive samples of SARS-CoV-2. Employing variant calling analysis on FASTQ reads and phylogenetic inference, we report a brief dispersion of a potentially new B.1.1.28-derived variant detected between 2021 May and June in individuals crossing the border between Brazil and Argentina, and local spread to inpatients from hospitals at the Rio Grande do Sul state capital (Porto Alegre). Besides, the Rio Grande do Sul State SARS-CoV-2 genomic epidemiological data was analyzed and showed an important B.1.1.28 peak in RS at the same period (May-June), even in the presence of a major Gamma wave. CONCLUSIONS: The emergence of a putative B.1.1.28-derived lineage was identified in travelers crossing Brazil-Argentina border representing an important peak of B.1.1.28 in RS State with a decreased in Gamma variant frequency in the same period of time.

Hepatitis E virus genotype 3 in bovine livers slaughtered in the state of Rio Grande do Sul, Brazil.

Foodborne viruses are becoming a global concern as they overwhelm the health system and have the potential to spread globally. Among them, some genotypes of hepatitis E virus (HEV), which is one of the main causes of acute hepatitis in humans, have a zoonotic potential and can be found in foods of animal origin. Infected farm animals are a possible source of the virus, either by direct contact with animal excreta or meat. In the present study, 240 bovine liver samples from slaughter carried out in Rio Grande do Sul (RS), Brazil, were analyzed and tested for the presence of HEV. After performing PCR, 5.4% of positive samples were observed. One of the samples could be identified by molecular phylogenetic analysis as belonging to genotype 3, for which pigs are natural reservoirs, but has not been reported in bovine meat and products so far.

Early introduction, dispersal and evolution of Delta SARS-CoV-2 in Southern Brazil, late predominance of AY.99.2 and AY.101 related lineages.

The emergence of Variants of Concern (VOC) presenting an unusual number of new mutations is one of the most remarkable features of SARS-CoV-2. The Delta variant, since its appearance, replaced the VOC Gamma, which was responsible for the major COVID-19 wave in Brazil. In this study, we performed a Delta whole-genome sequencing of 183 samples as part of a major genomic surveillance study performed since the beginning of the pandemic. Here, we showed an emergence, widespread dispersion and consolidation of the Delta variant in Rio Grande do Sul State, completely replacing the Gamma variant in a four to five months period. Performing the phylogenetic and phylodynamic analysis, the majority of the sequences generated herein were classified as AY.99.2, AY.99.2-like and AY.101. AY.99.2 Delta-related lineage has been widely reported in Brazil and in the Americas as well. Altogether, our findings provided a mutational profile of the sequences and presented high substitutions per site in the root-to-tip phylogenetic tree, corroborating studies that show the high mutational rate of SARS-CoV-2 over time.

Y380Q novel mutation in receptor-binding domain of SARS-CoV-2 spike protein together with C379W interfere in the neutralizing antibodies interaction.

We aimed to describe the SARS-CoV-2 lineages circulating early pandemic among samples with S gene dropout and characterize the receptor-binding domain (RBD) of viral spike protein. Adults and children older than 2 months with signs and symptoms of COVID-19 were prospectively enrolled from May to October in Porto Alegre, Brazil. All participants performed RT-PCR assay, and samples with S gene dropout and cycle threshold < 30 were submitted to high-throughput sequencing (HTS). 484 out of 1,557 participants tested positive for SARS-CoV-2. The S gene dropout was detected in 7.4% (36/484) and a peak was observed in August. The B.1.1.28, B.1.91 and B.1.1.33 lineages were circulating in early pandemic. The RBD novel mutation (Y380Q) was found in one sample occurring simultaneously with C379W and V395A, and the B.1.91 lineage in the spike protein. The Y380Q and C379W may interfere with the binding of neutralizing antibodies (CR3022, EY6A, H014, S304).

Predominance of SARS-CoV-2 P.1 (Gamma) lineage inducing the recent COVID-19 wave in southern Brazil and the finding of an additional S: D614A mutation.

Recently, the highest wave of SARS-CoV-2 epidemic occurred since the beginning of the pandemic in Brazil was registered in Rio Grande do Sul (RS) State, Southern Brazil, considering the number of cases, deaths and hospitalization per day caused by COVID-19. In this study we described which lineages were circulating in the first quarter of 2021 in Southern Brazil to better understand the viral factors involved in the health crisis caused by SARS-CoV-2 in the region, searching also for possible additional SARS-CoV-2 sequence mutations. A total of 70 positive SARS-CoV-2 samples collected between January 28th, 2021 until April 23rd, 2021, were selected to sequencing. Whole genome sequencing of 70 SARS-CoV-2 samples showed a predominance of Gamma lineage (67%, 47/70), followed by P.2 lineage (27%, 19/70) and B.1.1.28 (6%, 4/70). Two Gamma lineage consensus sequences presented a new S:D614A mutation. Newly mutations could be emerging due the quick SARS-CoV-2 spreading. Thus, the greater understanding about immune protection and variants vigilance is essential to the better management of the health SARS-CoV-2 crisis.

Reinfection cases by closely related SARS-CoV-2 lineages in Southern Brazil.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic that started in late 2019 and still affects people's lives all over the world. Lack of protective immunity after primary infection has been involved with reported reinfection cases by SARS-CoV-2. In this study, we described two cases of reinfection caused by non-VOC (Variants of Concern) strains in southern Brazil, being one patient a healthcare worker. The four samples previously positive for SARS-CoV-2 by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) were sequenced by a high-performance platform and the genomic analysis confirmed that lineages responsible for infections were B.1.91 and B.1.1.33 (patient 1), and B.1.1.33 and B.1.1.28 (patient 2). The interval between the two positive RT-qPCR for patients 1 and 2 was 45 and 61 days, respectively. This data shows that patients may be reinfected even by very closely related SARS-CoV-2 lineages.

Early detection of SARS-CoV-2 P.1 variant in Southern Brazil and reinfection of the same patient by P.2.

Multiple variants of the Severe Acute Respiratory Syndrome coronavirus 2 virus (SARS-CoV-2) have been constantly reported across the world. The B.1.1.28 lineage has been evolving in Brazil since February 2020 and originated the P.1 variant of concern (VOC), recently named as the Gamma variant by the newly WHO nomenclature proposal, and P.2 as a variant of interest (VOI). Here we describe an early case of P.1 primary infection in Southern Brazil in late November 2020, soon after the emergence of the variant in Manaus, Northern Brazil. The same male patient was reinfected by another B.1.1.28 variant, namely P.2, in March, 2021. The genomic analysis confirmed genetically significant differences between the two viruses recovered in both infections, the P.1 lineage in the first episode and P.2 in the reinfection. Due the very early detection of P.1, we have also investigated the circulation of P.1 in the same region by differential RT-qPCR, showing that this was an isolated case of P.1 at the time of detection, and this variant has disseminated and became prominent from late January to the end of March, 2021. SARS-CoV-2 recent reports of reinfection have raised critical questions on whether and how well a first infection protects against reinfection.

Genomic epidemiology of SARS-CoV-2 in Esteio, Rio Grande do Sul, Brazil.

BACKGROUND: Brazil is the third country most affected by Coronavirus disease-2019 (COVID-19), but viral evolution in municipality resolution is still poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We aimed to track molecular evolution and spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Esteio (Southern Brazil) using phylogenetics and phylodynamics inferences from 21 new genomes in global and regional context. Importantly, the case fatality rate (CFR) in Esteio (3.26%) is slightly higher compared to the Rio Grande do Sul (RS) state (2.56%) and the entire Brazil (2.74%). RESULTS: We provided a comprehensive view of mutations from a representative sampling from May to October 2020, highlighting two frequent mutations in spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in spike Receptor Binding Domain (RBD) characteristic of the B.1.351 and P.1 lineages, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). E484K was found in two genomes from mid-October, which is the earliest description of this mutation in Southern Brazil. Lineages containing this substitution must be subject of intense surveillance due to its association with immune evasion. We also found two epidemiologically-related clusters, including one from patients of the same neighborhood. Phylogenetics and phylodynamics analysis demonstrates multiple introductions of the Brazilian most prevalent lineages (B.1.1.33 and B.1.1.248) and the establishment of Brazilian lineages ignited from the Southeast to other Brazilian regions. CONCLUSIONS: Our data show the value of correlating clinical, epidemiological and genomic information for the understanding of viral evolution and its spatial distribution over time. This is of paramount importance to better inform policy making strategies to fight COVID-19.