Lack of molecular evidence of fecal-borne viruses in capybaras from São Paulo state, Brazil, 2018-2020: a minor public health issue.

Capybara (Hydrochoerus hydrochaeris) is the world's largest rodent species distributed throughout South America. These animals are incredibly tolerant to anthropogenic environments and are occupying large urban centers. Capybaras are known to carry potentially zoonotic agents, including R. rickettsia, Leishmania spp., Leptospira spp., Trypanosoma spp., Salmonella spp., Toxoplasma gondii, and rabies virus. Focusing on the importance of monitoring potential sources of emerging zoonotic viruses and new viral reservoirs, the aim of the present study was to assess the presence of fecal-borne viruses in the feces of capybaras living in urban parks in São Paulo state, Brazil. A total of 337 fecal samples were collected between 2018 and 2020 and screened for the following: (i) Rotavirus group A (RVA) by ELISA; (ii) non-RVA species and Picobirnavirus (PBV) using PAGE; (iii) Human Bocaparvovirus (HBoV), Bufavirus (BuV), Tusavirus (TuV), and Cutavirus (CuV) qPCR; (iv) Human Enterovirus (EV), Norovirus GII (NoV), and Hantavirus by in houses RT-qPCR; (v) SARS-CoV-2 via commercial RT-qPCR kit assay; and (vi) Astrovirus (AstV) and Adenovirus (AdV) using conventional nested (RT)-PCRs. All fecal samples tested were negative for fecal-borne viruses. This study adds further evidence that the fecal-borne viruses is a minor public health issue in Brazilian capybaras, at least during the surveillance period and surveyed areas. Continuous monitoring of sylvatic animals is essential to prevent and control the emergence or re-emergence of newly discovered virus as well as viruses with known zoonotic potential.

Discovery of novel Mamastroviruses in Bactrian camels and dromedaries reveals complex recombination history.

Virus emergence may occur through interspecies transmission and recombination of viruses coinfecting a host, with potential to pair novel and adaptive gene combinations. Camels are known to harbor diverse ribonucleic acid viruses with zoonotic and epizootic potential. Among them, astroviruses are of particular interest due to their cross-species transmission potential and endemicity in diverse host species, including humans. We conducted a molecular epidemiological survey of astroviruses in dromedaries from Saudi Arabia and Bactrian camels from Inner Mongolia, China. Herein, we deployed a hybrid sequencing approach coupling deep sequencing with rapid amplification of complementary deoxyribonucleic acid ends to characterize two novel Bactrian and eight dromedary camel astroviruses, including both partial and complete genomes. Our reported sequences expand the known diversity of dromedary camel astroviruses, highlighting potential recombination events among the astroviruses of camelids and other host species. In Bactrian camels, we detected partially conserved gene regions bearing resemblance to human astrovirus types 1, 4, and 8 although we were unable to recover complete reading frames from these samples. Continued surveillance of astroviruses in camelids, particularly Bactrian species and associated livestock, is highly recommended to identify patterns of cross-species transmission and to determine any epizootic threats and zoonotic risks posed to humans. Phylogenomic approaches are needed to investigate complex patterns of recombination among the astroviruses and to infer their evolutionary history across diverse host species.

A traveling SARS-CoV-2 laboratory as part of a pandemic response among vulnerable Brazilian populations.

BACKGROUND: Brazil has been dramatically hit by the SARS-CoV-2 pandemic and is a world leader in COVID-19 morbidity and mortality. Additionally, the largest country of Latin America has been a continuous source of SARS-CoV-2 variants and shows extraordinary variability of the pandemic strains probably related to the country´s outstanding position as a Latin American economical and transportation hub. Not all regions of the country show sufficient infrastructure for SARS-CoV-2 diagnosis and genotyping which can negatively impact the pandemic response. METHODS: Due to this reason and to disburden the diagnostic system of the inner São Paulo State, the Butantan Institute established the Mobile Laboratory (in Portuguese: LabMovel) for SARS-CoV-2 testing which started a trip of the most important "hotspots" of the most populous Brazilian region. The LabMovel initiated in two important cities of the State: Aparecida do Norte (an important religious center) and the Baixada Santista region which incorporates the port of Santos, the busiest in Latin America. The LabMovel was fully equipped with an automatized system for SARS-CoV-2 diagnosis and sequencing/genotyping. It also integrated the laboratory systems for patient records and results divulgation including in the Federal Brazilian Healthcare System. RESULTS: Currently,16,678 samples were tested, among them 1,217 from Aparecida and 4,564 from Baixada Santista. We tracked the delta introductio in the tested regions with its high diversification. The established mobile SARS-CoV-2 laboratory had a major impact on the Public Health System of the included cities including timely delivery of the results to the healthcare agents and the Federal Healthcare system, evaluation of the vaccination status of the positive individuals in the background of exponential vaccination process in Brazil and scientific and technological divulgation of the fieldwork to the most vulnerable populations. CONCLUSIONS: The SARS-CoV-2 pandemic has demonstrated worldwide the importance of science to fight against this viral agent and the LabMovel shows that it is possible to integrate researchers, clinicians, healthcare workers and patients to take rapid actions that can in fact mitigate this and other epidemiological situations.

Genomic surveillance unfolds the SARS-CoV-2 transmission and divergence dynamics in Bangladesh.

The highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4,622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7,659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure. Together with an ongoing genomic surveillance program, these data will contribute to a better understanding of SARS-CoV-2, as well as its evolution pattern and pandemic characteristics in Bangladesh.

Challenges and Opportunities for Global Genomic Surveillance Strategies in the COVID-19 Era.

Global SARS-CoV-2 genomic surveillance efforts have provided critical data on the ongoing evolution of the virus to inform best practices in clinical care and public health throughout the pandemic. Impactful genomic surveillance strategies generally follow a multi-disciplinary pipeline involving clinical sample collection, viral genotyping, metadata linkage, data reporting, and public health responses. Unfortunately, current limitations in each of these steps have compromised the overall effectiveness of these strategies. Biases from convenience-based sampling methods can obfuscate the true distribution of circulating variants. The lack of standardization in genotyping strategies and bioinformatic expertise can create bottlenecks in data processing and complicate interpretation. Limitations and inconsistencies in clinical and demographic data collection and sharing can slow the compilation and limit the utility of comprehensive datasets. This likewise can complicate data reporting, restricting the availability of timely data. Finally, gaps and delays in the implementation of genomic surveillance data in the public health sphere can prevent officials from formulating effective mitigation strategies to prevent outbreaks. In this review, we outline current SARS-CoV-2 global genomic surveillance methods and assess roadblocks at each step of the pipeline to identify potential solutions. Evaluating the current obstacles that impede effective surveillance can improve both global coordination efforts and pandemic preparedness for future outbreaks.

From Clinical Specimen to Whole Genome Sequencing of A(H3N2) Influenza Viruses: A Fast and Reliable High-Throughput Protocol.

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Respiratory Syncytial Virus: New Challenges for Molecular Epidemiology Surveillance and Vaccination Strategy in Patients with ILI/SARI.

Several respiratory pathogens are responsible for influenza-like illness (ILI) and severe respiratory infections (SARI), among which human respiratory syncytial virus (hRSV) represents one of the most common aetiologies. We analysed the hRSV prevalence among subjects with ILI or SARI during the five influenza seasons before the emergence of SARS-CoV-2 epidemic in Sicily (Italy). Respiratory specimens from ILI outpatients and SARI inpatients were collected in the framework of the Italian Network for the Influenza Surveillance and molecularly tested for hRSV-A and hRSV-B. Overall, 8.1% of patients resulted positive for hRSV. Prevalence peaked in the age-groups <5 years old (range: 17.6-19.1%) and ≥50 years old (range: 4.8-5.1%). While the two subgroups co-circulated throughout the study period, hRSV-B was slightly predominant over hRSV-A, except for the season 2019-2020 when hRSV-A strongly prevailed (82.9%). In the community setting, the distribution of hRSV subgroups was balanced (47.8% vs. 49.7% for hRSV-A and hRSV-B, respectively), while most infections identified in the hospital setting were caused by hRSV-B (69.5%); also, this latter one was more represented among hRSV cases with underlying diseases, as well as among those who developed a respiratory complication. The molecular surveillance of hRSV infections may provide a valuable insight into the epidemiological features of ILI/SARI. Our findings add new evidence to the existing knowledge on viral aetiology of ILI and SARI in support of public health strategies and may help to define high-risk categories that could benefit from currently available and future vaccines.

A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in SARS-CoV-2 surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.

Genomic Analysis and Lineage Identification of SARS-CoV-2 Strains in Migrants Accessing Europe Through the Libyan Route.

Many African countries, representing the origin of the majority of refugees, asylum-seekers, and other migrants, toward regions bordering on the Mediterranean area, are experiencing sustained local transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sicily is one of the main entry gates of migrants crossing into Europe. We conducted a pilot study, based on the full-genome sequencing of SARS-CoV-2 strains isolated from migrants coming to Sicily by crossing the Mediterranean Sea, with the aim to investigate the viral genome polymorphism and to describe their genetic variations and the phylogenetic relationships. On June 21, a nongovernmental organization vessel rescued 210 migrants crossing the Mediterranean Sea from Libya to Sicily. Of them, 13.4% tested positive for SARS-CoV-2. Eighteen whole genome sequences were obtained to explore viral genetic variability. All but one of the sequences clustered with other viral African strains within the lineage A, whereas only one intermixed among B.1 lineage genomes. Our findings documented that most of the investigated migrants acquired SARS-CoV-2 infection before landing in Sicily. However, SARS-CoV-2 transmission during travel or in overcrowded Libyan immigrant camps and/or illegal transport boats could not be ruled out. SARS-CoV-2 molecular surveillance on migrants arriving in Europe through the Sicilian gate may improve the knowledge of global SARS-CoV-2 transmission dynamic also in light of the emergence of new variants.

Surveillance of SARS-CoV-2 in Frankfurt am Main from October to December 2020 Reveals High Viral Diversity Including Spike Mutation N501Y in B.1.1.70 and B.1.1.7.

BACKGROUND: International travel is a major driver of the introduction and spread of SARS-CoV-2. AIM: To investigate SARS-CoV-2 genetic diversity in the region of a major transport hub in Germany, we characterized the viral sequence diversity of the SARS-CoV-2 variants circulating in Frankfurt am Main, the city with the largest airport in Germany, from the end of October to the end of December 2020. METHODS: In total, we recovered 136 SARS-CoV-2 genomes from nasopharyngeal swab samples. We isolated 104 isolates that were grown in cell culture and RNA from the recovered viruses and subjected them to full-genome sequence analysis. In addition, 32 nasopharyngeal swab samples were directly sequenced. RESULTS AND CONCLUSION: We found 28 different lineages of SARS-CoV-2 circulating during the study period, including the variant of concern B.1.1.7 (Δ69/70, N501Y). Six of the lineages had not previously been observed in Germany. We detected the spike protein (S) deletion Δ69/Δ70 in 15% of all sequences, a four base pair (bp) deletion (in 2.9% of sequences) and a single bp deletion (in 0.7% of sequences) in ORF3a, leading to ORF3a truncations. In four sequences (2.9%), an amino acid deletion at position 210 in S was identified. In a single sample (0.7%), both a 9 bp deletion in ORF1ab and a 7 bp deletion in ORF7a were identified. One sequence in lineage B.1.1.70 had an N501Y substitution while lacking the Δ69/70 in S. The high diversity of sequences observed over two months in Frankfurt am Main highlights the persisting need for continuous SARS-CoV-2 surveillance using full-genome sequencing, particularly in cities with international airport connections.

Molecular Epidemiology Surveillance of SARS-CoV-2: Mutations and Genetic Diversity One Year after Emerging.

In December 2019, the first cases of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified in the city of Wuhan, China. Since then, it has spread worldwide with new mutations being reported. The aim of the present study was to monitor the changes in genetic diversity and track non-synonymous substitutions (dN) that could be implicated in the fitness of SARS-CoV-2 and its spread in different regions between December 2019 and November 2020. We analyzed 2213 complete genomes from six geographical regions worldwide, which were downloaded from GenBank and GISAID databases. Although SARS-CoV-2 presented low genetic diversity, there has been an increase over time, with the presence of several hotspot mutations throughout its genome. We identified seven frequent mutations that resulted in dN substitutions. Two of them, C14408T>P323L and A23403G>D614G, located in the nsp12 and Spike protein, respectively, emerged early in the pandemic and showed a considerable increase in frequency over time. Two other mutations, A1163T>I120F in nsp2 and G22992A>S477N in the Spike protein, emerged recently and have spread in Oceania and Europe. There were associations of P323L, D614G, R203K and G204R substitutions with disease severity. Continuous molecular surveillance of SARS-CoV-2 will be necessary to detect and describe the transmission dynamics of new variants of the virus with clinical relevance. This information is important to improve programs to control the virus.