Zika virus infections and suspected microcephaly cases have been reported in Angola since late 2016, but no data are available about the origins, epidemiology, and diversity of the virus. We aimed to investigate the emergence and circulation of Zika virus in Angola.

Genomic epidemiology can provide a unique, real-time understanding of SARS-CoV-2 transmission patterns. Yet the potential for genomic analyses to guide local policy and community-based behavioral decisions is limited because they are often oriented towards specially trained scientists and conducted on a national or global scale. Here, we propose a new paradigm: Phylogenetic analyses performed on a local level (municipal, county, or state), with results communicated in a clear, timely, and actionable manner to strengthen public health responses. We believe that presenting results rapidly, and tailored to a non-expert audience, can serve as a template for effective public health response to COVID-19 and other emerging viral diseases.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

The characteristics of pneumococcal carriage vary between infants and adults. Host immune factors have been shown to contribute to these age-specific differences, but the role of pathogen sequence variation is currently less well-known. Identification of age-associated pathogen genetic factors could leadto improved vaccine formulations. We therefore performed genome sequencing in a large carriage cohort of children and adults and combined this with data from an existing age-stratified carriage study. We compiled a dictionary of pathogen genetic variation, including serotype, strain, sequence elements, single-nucleotide polymorphisms (SNPs), and clusters of orthologous genes (COGs) for each cohort - all of which were used in a genome-wide association with host age. Age-dependent colonization showed weak evidence of being heritable in the first cohort (h2 = 0.10, 95% CI 0.00-0.69) and stronger evidence in the second cohort (h2 = 0.56, 95% CI 0.23-0.87). We found that serotypes and genetic background (strain) explained a proportion of the heritability in the first cohort (h2serotype = 0.07, 95% CI 0.04-0.14 and h2GPSC = 0.06, 95% CI 0.03-0.13) and the second cohort (h2serotype = 0.11, 95% CI 0.05-0.21 and h2GPSC = 0.20, 95% CI 0.12-0.31). In a meta-analysis of these cohorts, we found one candidate association (p=1.2 × 10-9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, while we did find a small effect of pathogen genome variation on pneumococcal carriage between child and adult hosts, this was variable between populations and does not appear to be caused by strong effects of individual genes. This supports proposals for adaptive future vaccination strategies that are primarily targeted at dominant circulating serotypes and tailored to the composition of the pathogen populations.

Pneumococcus is exposed to a variety of temperature and oxygen levels in the upper respiratory tract and as it invades the lung, tissues, and blood. We sought to determine the effect of environmental variability on growth in vitro and to assess variability between strains. We evaluated the effect of temperature and oxygen on the growth of 256 isolates representing 53 serotypes, recovered from healthy carriers and disease patients. Strains were grown at a range of temperatures, anaerobically or in ambient air with catalase, and were monitored by reading the optical density. Regression models evaluated variation in the characteristics of the growth curves.

Since its emergence and detection in Wuhan, China in late 2019, the novel coronavirus SARS-CoV-2 has spread to nearly every country around the world, resulting in hundreds of thousands of infections to date. The virus was first detected in the Pacific Northwest region of the United States in January, 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the U.S., we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated impacts of federal travel restrictions. This study provides evidence for widespread, sustained transmission of SARS-CoV-2 within the U.S. and highlights the critical need for local surveillance.

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

This study aimed to investigate the characteristics related to SARS-CoV-2 in Luanda, Angola. A total of 622 individuals were screened for SARS-CoV-2 from January to September 2020. Chi-square and logistic regression were used to identify the relationship between sociodemographic characteristics and SARS-CoV-2. Of the 622 tested, 14.3% tested positive. The infection rate was the same for both genders (14.3%). Individuals ≥40 years old, from non-urbanized areas, and healthcare professionals had a higher frequency of infection. The risk of infection was very high in individuals ≥60 years old (AOR: 23.3, 95% CI: 4.83-112), in women (AOR: 1.24, 95% CI: 0.76-2.04), in Luanda (AOR: 7.40, 95% CI: 1.64-33.4), and healthcare professionals (AOR: 1.27, 95% CI: 0.60-2.71), whereas a low risk was observed in individuals from urbanized areas (AOR: 0.44, 95% CI: 0.26-0.75). Our results suggest that Angolan authorities should implement a greater effort in non-urbanized areas and among healthcare professionals since when these individuals presented any indication for a COVID-19 test, such as fever/cough/myalgia, they were more likely to test positive for SARS-CoV-2 than having some other cause for symptoms.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

No abstract available

Genomic sequencing is crucial to understanding the epidemiology and evolution of SARS-CoV-2. Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal swabs, as input into whole genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays, however saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from nasopharyngeal swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals, like HCW.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.

Monitoring genetic diversity and drug resistance mutations (DRMs) is critical for understanding HIV epidemiology. Here, we report HIV-1 genetic diversity and DRMs in blood samples from 42 HIV-positive pregnant women naive to antiretroviral therapy (ART), in Luanda. The samples were subjected to nested-PCR, followed by sequencing of HIV-1 pol gene, targeting the protease and reverse transcriptase fragments. HIV-1 diversity was analyzed using the REGA HIV-1 subtyping tool and DRMs were identified using the Calibrated Population Resistance tool. A total of 34 sequences were obtained. The data revealed wide HIV-1 subtypes heterogeneity, with subtype C (38%, 13/34) the most frequent, followed by the subtypes F1 (18%, 6/34), A1 (9%, 3/34), G (9%, 3/34), D (6%, 2/34) and H (3%, 1/34). In addition, recombinants strains were detected, with CRF02_AG (6%, 2/34) the most frequent, followed by CRF37_cpx, F1/C, A1/G and H/G, all with 3% (1/34). A total of 6/34 (18%) of the sequences presented DRMs. The non-nucleoside reverse transcriptase inhibitors presented 15% (5/34) of resistance. Moreover, 1/34 (3%) sequence presented resistance against both non-nucleoside reverse transcriptase inhibitors and nucleoside reverse transcriptase inhibitors, simultaneously. Despite the small sample size, our results suggest the need to update currently used ART regimens. Surveillance of HIV-1 subtypes and DRMs are necessary to understand HIV epidemiology and to guide modification of ART guidelines in Angola.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se , but rather with the delayed kinetics of NAb production.

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates.

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause life-threatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, identifying variants in CCDC33 associated with susceptibility. Pneumococcal genetic variation explains a large amount of invasive potential (70%), but has no effect on severity. Serotype alone is insufficient to explain invasiveness, suggesting other pneumococcal factors are involved in progression to invasive disease. We identify pneumococcal genes involved in invasiveness including pspC and zmpD, and perform a human-bacteria interaction analysis. These genes are potential candidates for the development of more broadly-acting pneumococcal vaccines.