Occurrences of similar viral diversity in campus wastewater and reclaimed water of a university dormitory.

Water reuse from wastewater sources still remain some critical safety concerns associated with treacherous contaminants like pathogenic viruses. In this study, viral diversities in campus wastewater (CWW) and its reclaimed water (RCW) recycled for toilet flushing and garden irrigation of a university dormitory were assessed using metagenomic sequencing for acquisition of more background data. Results suggested majority (>80%) of gene sequences within assembled contigs predicted by open reading frame (ORF) finder were no-hit yet believed to be novel/unrevealed viral genomic information whereas hits matched bacteriophages (i.e., mainly Myoviridae, Podoviridae, and Siphoviridae families) were predominant in both CWW and RCW samples. Moreover, few pathogenic viruses (<1%) related to infections of human skin (e.g., Molluscum contagiosum virus, MCV), digestion system (e.g., hepatitis C virus, HCV), and gastrointestinal tract (e.g., human norovirus, HuNoV) were also noticed raising safety concerns about application of reclaimed waters. Low-affinity interactions of particular viral exterior proteins (e.g., envelope glycoproteins or spike proteins) for disinfectant ligand (e.g., chlorite) elucidated treatment limitations of current sewage processing systems even with membrane bioreactor and disinfectant contactor. Revolutionary disinfection approaches together with routine monitoring and new regulations are prerequisite to secure pathogen-correlated water quality for safer reuse of reclaimed waters.

Virome diversity of Hyalomma dromedarii ticks collected from camels in the United Arab Emirates.

Background and Aim: Viruses are important components of the microbiome of ticks. Ticks are capable of transmitting several serious viral diseases to humans and animals. Hitherto, the composition of viral communities in Hyalomma dromedarii ticks associated with camels in the United Arab Emirates (UAE) remains unexplored. This study aimed to characterize the RNA virome diversity in male and female H. dromedarii ticks collected from camels in Al Ain, UAE. Materials and Methods: We collected ticks, extracted, and sequenced RNA, using Illumina (NovaSeq 6000) and Oxford Nanopore (MinION). Results: From the total generated sequencing reads, 180,559 (~0.35%) and 197,801 (~0.34%) reads were identified as virus-related reads in male and female tick samples, respectively. Taxonomic assignment of the viral sequencing reads was accomplished based on bioinformatic analyses. Further, viral reads were classified into 39 viral families. Poxiviridae, Phycodnaviridae, Phenuiviridae, Mimiviridae, and Polydnaviridae were the most abundant families in the tick viromes. Notably, we assembled the genomes of three RNA viruses, which were placed by phylogenetic analyses in clades that included the Bole tick virus. Conclusion: Overall, this study attempts to elucidate the RNA virome of ticks associated with camels in the UAE and the results obtained from this study improve the knowledge of the diversity of viruses in H. dromedarii ticks.

Rise and fall of SARS-CoV-2 variants in Rotterdam: Comparison of wastewater and clinical surveillance.

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity.

The Prevalence of Viral Pathogens among Bats in Kazakhstan.

Bats carry thousands of viruses from 28 different families. To determine the presence of various pathogens in bat populations in Kazakhstan, 1149 samples (393 oropharyngeal swabs, 349 brain samples, 407 guano) were collected. The samples were collected from four species of bats (Vespertilio murinus, Nyctalus noctula, Myotis blythii, Eptesicus serotinus) in nine regions. The Coronavirus RNA was found in 38 (4.75%) samples, and the rabies virus in 27 (7.74%) samples from bats. Coronaviruses and the rabies virus were found in bats in six out of nine studied areas. The RNAs of SARS-CoV-2, MERS, TBE, CCHF, WNF, influenza A viruses were not detected in the bat samples. The phylogeny of the RdRp gene of 12 samples made it possible to classify them as alphacoronaviruses and divide them into two groups. The main group (n = 11) was closely related to bat coronaviruses from Ghana, Zimbabwe and Kenya. The second group (n = 1) was closely related to viruses previously isolated in the south of Kazakhstan. The phylogeny of the N gene sequence from a bat from west Kazakhstan revealed its close relationship with isolates from the Cosmopolitan group of rabies viruses (Central Asia). These results highlight the need for a continuous monitoring of volatile populations to improve the surveillance and detection of infectious diseases.

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses.

Next generation sequencing (NGS)/deep sequencing has become an important tool in the study of viruses. The use of unique molecular identifiers (UMI) can overcome the limitations of PCR errors and PCR-mediated recombination and reveal the true sampling depth of a viral population being sequenced in an NGS experiment. This approach of enhanced sequence data represents an ideal tool to study both high and low abundance drug resistance mutations and more generally to explore the genetic structure of viral populations. Central to the use of the UMI/Primer ID approach is the creation of a template consensus sequence (TCS) for each genome sequenced. Here we describe a series of experiments to validate several aspects of the Multiplexed Primer ID (MPID) sequencing approach using the MiSeq platform. We have evaluated how multiplexing of cDNA synthesis and amplicons affects the sampling depth of the viral population for each individual cDNA and amplicon to understand the relationship between broader genome coverage versus maximal sequencing depth. We have validated reproducibility of the MPID assay in the detection of minority mutations in viral genomes. We have also examined the determinants that allow sequencing reads of PCR recombinants to contaminate the final TCS data set and show how such contamination can be limited. Finally, we provide several examples where we have applied MPID to analyze features of minority variants and describe limits on their detection in viral populations of HIV-1 and SARS-CoV-2 to demonstrate the generalizable utility of this approach with any RNA virus.

HIV-1 DIAGNOSES in NORTH CAROLINA, 2018-21: INCIDENT INFECTIONS and DRUG RESISTANCE

Background: Knowing the true incidence of HIV-1 infections (recent infections) among people newly diagnosed is pivotal to monitoring the course of the epidemic. We have developed a Primer ID Next Gen Sequencing (PID-NGS) assay to identify recent infection by measuring within-host viral diversity over multiple regions of the HIV-1 genome. We implemented a state-wide project to identify recent infections and transmitted drug resistance mutations (DRMs) in diagnostic samples in near real time. Methods: Serum samples from individuals with newly HIV-1 diagnoses (diagnostic sample collected within 30 days of diagnosis) were sequenced. PID-NGS libraries were constructed covering the coding regions for protease, a portion of reverse transcriptase, integrase, and the env gene. The use of the PID-NGS strategy allows for significant error correction and also a definition of the sampling depth of the viral population. Recent infection was defined as within 9-month of infection. DRMs were summarized at detection sensitivities of 30%, 10% and 1% based on viral population sampling depth. Results: From Jan 2018 to Jun 2021, we successfully sequenced partial genomes from 743 individuals with new diagnoses. Year 2020 had the lowest number of new diagnoses (Fig 1a, red bar). Overall, 39.2% of samples were inferred to have represented infection within the previous 9 months. Percent of recent infection varied significantly over the years, increasing from 29.6% in late 2018 to 50.9% in early 2020, but decreasing significantly to 32.7% in 2021 (Fig 1a, blue lines). Individuals younger than 30 y/o were more likely to be identified with recent infection (p<0.01). NNRTI DRMs, especially K103N, were the most abundant DRMs. Fig 1b shows the trend of DRMs over the four years. We observed a trend of decrease in the overall NNRTI DRMs and an increase in the NRTI DRMs in the population. Further analysis suggests that the increase in NRTI DRMs were from TAMs and their revertants, while clinically important NRTI DRMs (K65R and M184) were low (<1%). Conclusion: We have demonstrated a state-wide, all-in-one platform to monitor HIV-1 recency and DRMs in new diagnoses. The number of new diagnoses decreased significantly in 2020 in concert with the COVID-19 pandemic which suggests a decrease in overall HIV testing. The decline in the percentage of recent infections in early 2021 signals a return to broader HIV-1 testing and diagnosis. The increase of other NRTI DRMs suggests ongoing evolution at these sites within the viral population.

MUTATIONS in PERSISTENT SARS-CoV-2 CASES

Background: The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies are of clinical importance. While several demographic and clinical correlates of Coronavirus Disease 2019 (COVID-19) outcome have been identified, their relationship to virological and immunological parameters remain poorly defined. Here, we evaluate viral diversity and the accumulation of intra-host mutations over time in a population of hospitalized adults positive for SARS-CoV-2. Methods: We performed longitudinal collection of nasopharyngeal swabs and blood samples from a small cohort of hospitalized adults with COVID-19. Clinical information regarding study subject's immunocompromised status was collected. Samples were assessed for SARS-CoV-2 viral load, viral genotype, viral diversity, and antibody titer. Results: Intra-host viral genetic diversity remained constant through disease course in study subjects that were non-immunocompromised and resulted in changes in viral genotype in some participants. We report the de novo emergence of Spike mutations that have been previously associated with circulating variants of concern in two immunosuppressed patients with persistent SARS-CoV-2 infection. Conclusion: Constant rates of viral evolution suggest the emergence of variants as a function of time, emphasizing the need for effective antivirals to control viral load over long disease courses.

SYMPTOM DURATION in COVID-19 CONVALESCENT PATIENTS: REGIONAL & CLINICAL ASSOCIATIONS

Background: Post-Acute Sequelae of SARS-CoV-2 (PASC) is characterized by persistent symptoms negatively impacting quality of life several weeks after SARS-CoV-2 diagnosis. Proposed risk factors include older age, female sex, comorbidities, and severe COVID-19, including hospitalization and oxygen requirement. Yet, associations of these factors with prolonged symptoms remain poorly understood globally. Methods: The global, observational cohort study HVTN 405/HPTN 1901 characterizes the clinical and immunologic course in the first year after SARS-CoV-2 infection among adults. The cohort was categorized by infection severity (asymptomatic;symptomatic with no oxygen requirement [NOR];non-invasive oxygen requirement [NIOR];or invasive oxygen requirement [IOR]). A regression model was applied to estimate geometric mean ratios (GMR) for duration and odds ratios (OR) for persistence of symptoms. Results: 759 participants from Peru (25.2%), USA (26.0%), Republic of South Africa (RSA, 37.7%), and non-RSA Sub-Saharan Africa (11.2%) were enrolled a median of 51 (IQR 35-66) days post-diagnosis, from May 2020 to Mar 2021. 53.8% were female, 69.8% were <55yo (median 44yo, IQR 33-58) and identified as non-Hispanic Black (42.7%), Hispanic (27.9%) or non-Hispanic White (15.8%). Comorbidities included obesity (42.8%), hypertension (24%), diabetes (14%), HIV infection (11.6%) and lung disease (7.5%). 76.2% were symptomatic (NOR 47.4%;NIOR 22.9%;and IOR 5.8%). Among symptomatic participants, median acute COVID-19 duration was 20 days (IQR 11-35);43.3% had ≥1 persistent symptom after COVID-19 resolution (39.8% NOR;49.1 % NIOR+IOR;p=0.037);16.8% reported ≥1 symptom >42 days (14.0% NOR;21.6% NIOR+IOR;p=0.025). Symptom duration was not associated with age or sex assigned at birth but was associated with disease severity (GMR 2.09;95%CI 1.5-2.91, p<0.001 for NIOR vs NOR;not significant for IOR vs NIOR), lung disease (GMR 2.43;95%CI 1.42-4.16, p=0.001), and global region (p<0.05, see Figure 1). Prolonged viral shedding was associated with persistent diarrhea (OR 6.59;95%CI 1.65-26.86;p=0.008). Conclusion: A recovery course consistent with PASC was significantly associated with infection severity, lung disease, and region. Regional differences in symptom profiles and duration may be influenced by viral diversity, genetic, or cultural factors and likely reflect disparities in healthcare access and interventions. Better understanding PASC associations may improve clinical assessment and management globally.

The International Virus Bioinformatics Meeting 2022.

The International Virus Bioinformatics Meeting 2022 took place online, on 23-25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus-host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting.

Genetic Analysis of SARS-CoV-2 Variants in Mexico during the First Year of the COVID-19 Pandemic.

During the first year of the SARS-CoV-2 pandemic in Mexico, more than two million people were infected. In this study, we analyzed full genome sequences from 27 February 2020 to 28 February 2021 to characterize the geographical and temporal distribution of SARS-CoV-2 lineages and identify the most common circulating lineages during this period. We defined six different geographical regions with particular dynamics of lineage circulation. The Northeast and Northwest regions were the ones that exhibited the highest lineage diversity, while the Central south and South/Southeast regions presented less diversity with predominance of a certain lineage. Additionally, by late February 2021, lineage B.1.1.519 represented more than 89% of all circulating lineages in the country.

The International Virus Bioinformatics Meeting 2020.

The International Virus Bioinformatics Meeting 2020 was originally planned to take place in Bern, Switzerland, in March 2020. However, the COVID-19 pandemic put a spoke in the wheel of almost all conferences to be held in 2020. After moving the conference to 8-9 October 2020, we got hit by the second wave and finally decided at short notice to go fully online. On the other hand, the pandemic has made us even more aware of the importance of accelerating research in viral bioinformatics. Advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks. The International Virus Bioinformatics Meeting 2020 has attracted approximately 120 experts in virology and bioinformatics from all over the world to join the two-day virtual meeting. Despite concerns being raised that virtual meetings lack possibilities for face-to-face discussion, the participants from this small community created a highly interactive scientific environment, engaging in lively and inspiring discussions and suggesting new research directions and questions. The meeting featured five invited and twelve contributed talks, on the four main topics: (1) proteome and RNAome of RNA viruses, (2) viral metagenomics and ecology, (3) virus evolution and classification and (4) viral infections and immunology. Further, the meeting featured 20 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting.