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.

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.

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.

Corona- and Paramyxoviruses in Bats from Brazil: A Matter of Concern?

Chiroptera are one of the most diverse mammal orders. They are considered reservoirs of main human pathogens, where coronaviruses (CoVs) and paramyxoviruses (PMVs) may be highlighted. Moreover, the growing number of publications on CoVs and PMVs in wildlife reinforces the scientific community's interest in eco-vigilance, especially because of the emergence of important human pathogens such as the SARS-CoV-2 and Nipha viruses. Considering that Brazil presents continental dimensions, is biologically rich containing one of the most diverse continental biotas and presents a rich biodiversity of animals classified in the order Chiroptera, the mapping of CoV and PMV genetics related to human pathogens is important and the aim of the present work. CoVs can be classified into four genera: Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus. Delta- and gammacoronaviruses infect mainly birds, while alpha- and betacoronaviruses contain important animal and human pathogens. Almost 60% of alpha- and betacoronaviruses are related to bats, which are considered natural hosts of these viral genera members. The studies on CoV presence in bats from Brazil have mainly assayed phyllostomid, molossid and vespertilionid bats in the South, Southeast and North territories. Despite Brazil not hosting rhinophilid or pteropodid bats, which are natural reservoirs of SARS-related CoVs and henipaviruses, respectively, CoVs and PMVs reported in Brazilian bats are genetically closely related to some human pathogens. Most works performed with Brazilian bats reported alpha-CoVs that were closely related to other bat-CoVs, despite a few reports of beta-CoVs grouped in the Merbecovirus and Embecovirus subgenera. The family Paramyxoviridae includes four subfamilies (Avulavirinae, Metaparamyxovirinae, Orthoparamyxovirinae and Rubulavirinae), and bats are significant drivers of PMV cross-species viral transmission. Additionally, the studies that have evaluated PMV presence in Brazilian bats have mainly found sequences classified in the Jeilongvirus and Morbillivirus genera that belong to the Orthoparamyxovirinae subfamily. Despite the increasing amount of research on Brazilian bats, studies analyzing these samples are still scarce. When surveying the representativeness of the CoVs and PMVs found and the available genomic sequences, it can be perceived that there may be gaps in the knowledge. The continuous monitoring of viral sequences that are closely related to human pathogens may be helpful in mapping and predicting future hotspots in the emergence of zoonotic agents.

PCV3-associated reproductive failure in pig herds in Brazil.

Porcine circovirus type 3 (PCV3) has been widely detected worldwide in healthy and sick pigs. Recently its association with clinical disease and reproductive failure has been proven through the detection of intralesional viral mRNA in affected pigs. This study aims to describe the occurrence of PCV3-associated reproductive failure (abortions) in sow herds in southern Brazil. Eleven fetuses from five different litters from two herds were analyzed. These herds reported an increase in the rate of late-gestation abortions, stillbirths, and the percentage of mummified piglets. At gross examination, six of the fetuses had large caudally rotated ears and one fetus was mummified. Microscopically, multisystemic vasculitis, lymphocytic interstitial pneumonia, myocarditis, and encephalitis were observed. These six fetuses with gross and histological lesions were positive in qPCR analysis for PCV3, and PCV3 transcription was shown through in situ hybridization (ISH-RNA) within the histologic lesions. Samples from all 11 fetuses tested negative in PCR exam for Porcine Circovirus type 1 and 2, Porcine Reproductive and Respiratory Syndrome, Porcine Parvovirus, and Atypical Porcine Pestivirus. Furthermore, based on the ORF2 analysis, the PCV3a clade was identified. This is the first report of PCV3a-associated reproductive failure in pig herds in South America.

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.

The virome of the white-winged vampire bat Diaemus youngi is rich in circular DNA viruses.

In the Neotropical region, the white-winged vampire bat (Diaemus youngi) is the rarest of the three species of vampire bats. This bat species feeds preferentially on bird blood, and there is limited information on the viruses infecting D. youngi. Hence, this study aimed to expand the knowledge about the viral diversity associated with D. youngi by sampling and pooling the lungs, liver, kidneys, heart, and intestines of all animals using high-throughput sequencing (HTS) on the Illumina MiSeq platform. A total of three complete and 10 nearly complete circular virus genomes were closely related to gemykrogvirus (Genomoviridae family), smacovirus (Smacoviridae family), and torque teno viruses (TTVs) (Anelloviridae family). In addition, three sequences of bat paramyxovirus were detected and found to be closely related to viruses reported in Pomona roundleaf bats and rodents. The present study provides a snapshot of the viral diversity associated with white-winged vampire bats and provides a baseline for comparison to viruses detected in future outbreaks.

Temporal analysis of bovine pestivirus diversity in Brazil.

In this study, phylogenetic and evolutionary analyses of cattle pestiviruses (BVDV-1, 2 and HoBiPeV) originating in Brazil were used to investigate the temporal diversification of subgenotypes in the country. Inferred dated phylogeny and time of the most recent common ancestor (tMRCA) demonstrated that some BVDV subgenotypes (1a, 1b, 1d, 1e, and 2b) and HoBi-like sequences clustered according to the region in which they were collected and that the diversification of subgenotypes appears to have occurred around the introduction of first Bos taurus and then Bos indicus, followed by expansion to form the adapted Brazilian breeds. The present results help to elucidate the temporal facts that led to diversification of ruminant pestiviruses in cattle in Brazil.

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).

A putative PCV3-associated disease in piglets from Southern Brazil.

Porcine circovirus type 3 (PCV3) is widely distributed worldwide, and its association with clinical disease in pigs has been studied in recent years. This study describes a novel PCV3-associated clinical disease in piglets from Brazil. Since September 2020, we received 48 piglets with large caudally rotated ears, weakness, and dyspnea. Most piglets were from gilts and died 1-5 days after birth. Two piglets that presented similar clinical signs and survived until 35-60 days had a marked decrease in growth rate. At post-mortem examination, the lungs did not collapse due to marked interlobular edema. Microscopically, the main feature was multisystemic vasculitis characterized by lymphocytes and plasma cells infiltrating and disrupting the wall of vessels, lymphohistiocytic interstitial pneumonia, myocarditis, and encephalitis. Viral replication was confirmed in these lesions through in situ hybridization (ISH-RNA). Seventeen cases were positive for PCV3 in PCR analysis, and all samples tested negative for porcine circovirus (PCV1, and PCV2); porcine parvovirus (PPV1, 2, 5, and 6); atypical porcine pestivirus (APPV); porcine reproductive and respiratory syndrome (PRRSV); and ovine herpesvirus-2 (OvHV-2). Phylogenetic analysis of the ORF2 sequence from five different pig farms showed that the PCV3a clade is circulating among Brazil's swineherds and causing neonatal piglet losses. This is the first report of PCV3a-associated disease in neonatal pigs from farms in Brazil.

Detection of coronavirus in vampire bats (Desmodus rotundus) in southern Brazil.

The vampire bat (Desmodus rotundus) is a haematophagous animal that feeds exclusively on the blood of domestic mammals. Vampire bat feeding habits enable their contact with mammalian hosts and may enhance zoonotic spillover. Moreover, they may carry several pathogenic organisms, including coronaviruses (CoVs), for which they are important hosts. The human pathogens that cause severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) and possibly coronavirus disease 2019 (SARS-CoV-2) all originated in bats but required bridge hosts to spread into human populations. To monitor the presence of potential zoonotic viruses in bats, the present work evaluated the presence of CoVs in vampire bats from southern Brazil. A total of 101 vampire bats were captured and euthanized between 2017 and 2019 in Rio Grande do Sul state, southern Brazil. The brain, heart, liver, lungs, kidneys and intestines were collected and macerated individually. The samples were pooled and submitted to high-throughput sequencing (HTS) using the Illumina MiSeq platform and subsequently individually screened using a pancoronavirus RT-PCR protocol. We detected CoV-related sequences in HTS, but only two (2/101; 1.98%) animals had CoV detected in the intestines by RT-PCR. Partial sequences of RdRp and spike genes were obtained in the same sample and the RdRp region in the other sample. The sequences were classified as belonging to Alphacoronavirus. The sequences were closely related to alphacoronaviruses detected in vampire bats from Peru. The continuous monitoring of bat CoVs may help to map and predict putative future zoonotic agents with great impacts on human health.

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.