Enteric viruses replicate in salivary glands and infect through saliva.

Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.

Within-host evolutionary dynamics and tissue compartmentalization during acute SARS-CoV-2 infection.

The global evolution of SARS-CoV-2 depends in part upon the evolutionary dynamics within individual hosts with varying immune histories. To characterize the within-host evolution of acute SARS-CoV-2 infection, we sequenced saliva and nasal samples collected daily from vaccinated and unvaccinated individuals early during infection. We show that longitudinal sampling facilitates high-confidence genetic variant detection and reveals evolutionary dynamics missed by less-frequent sampling strategies. Within-host dynamics in both unvaccinated and vaccinated individuals appeared largely stochastic; however, in rare cases, minor genetic variants emerged to frequencies sufficient for forward transmission. Finally, we detected significant genetic compartmentalization of viral variants between saliva and nasal swab sample sites in many individuals. Altogether, these data provide a high-resolution profile of within-host SARS-CoV-2 evolutionary dynamics.IMPORTANCEWe detail the within-host evolutionary dynamics of SARS-CoV-2 during acute infection in 31 individuals using daily longitudinal sampling. We characterized patterns of mutational accumulation for unvaccinated and vaccinated individuals, and observed that temporal variant dynamics in both groups were largely stochastic. Comparison of paired nasal and saliva samples also revealed significant genetic compartmentalization between tissue environments in multiple individuals. Our results demonstrate how selection, genetic drift, and spatial compartmentalization all play important roles in shaping the within-host evolution of SARS-CoV-2 populations during acute infection.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Rapid self-test of unprocessed viruses of SARS-CoV-2 and its variants in saliva by portable wireless graphene biosensor.

We have developed a DNA aptamer-conjugated graphene field-effect transistor (GFET) biosensor platform to detect receptor-binding domain (RBD), nucleocapsid (N), and spike (S) proteins, as well as viral particles of original Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus and its variants in saliva samples. The GFET biosensor is a label-free, rapid (≤20 min), ultrasensitive handheld wireless readout device. The limit of detection (LoD) and the limit of quantitation (LoQ) of the sensor are 1.28 and 3.89 plaque-forming units (PFU)/mL for S protein and 1.45 and 4.39 PFU/mL for N protein, respectively. Cognate spike proteins of major variants of concern (N501Y, D614G, Y453F, Omicron-B1.1.529) showed sensor response ≥40 mV from the control (aptamer alone) for fM to nM concentration range. The sensor response was significantly lower for viral particles and cognate proteins of Middle East Respiratory Syndrome (MERS) compared to SARS-CoV-2, indicating the specificity of the diagnostic platform for SARS-CoV-2 vs. MERS viral proteins. During the early phase of the pandemic, the GFET sensor response agreed with RT-PCR data for oral human samples, as determined by the negative percent agreement (NPA) and positive percent agreement (PPA). During the recent Delta/Omicron wave, the GFET sensor also reliably distinguished positive and negative clinical saliva samples. Although the sensitivity is lower during the later pandemic phase, the GFET-defined positivity rate is in statistically close alignment with the epidemiological population-scale data. Thus, the aptamer-based GFET biosensor has a high level of precision in clinically and epidemiologically significant SARS-CoV-2 variant detection. This universal pathogen-sensing platform is amenable for a broad range of public health applications and real-time environmental monitoring.

A shedding analysis after AAV8 CNS injection revealed fragmented viral DNA without evidence of functional AAV particles in mice.

Adeno-associated viruses (AAV) are commonly used in the scientific field due to their diverse application range. However, AAV shedding, the release of virions from the host organism, can impact the safety of AAV-based approaches. An increasing number of authorities require the characterization of vector shedding in clinical trials. Recently, shedding of transduced laboratory animals has also gained attention regarding the necessary disposal measures of their waste products. However, no explicit international regulations for AAV-shedding waste exist. Generating insights into shedding dynamics becomes increasingly relevant to help authorities develop adequate regulations. To date, knowledge of AAV vector shedding in mice is very limited. Moreover, confirmation of functional shed AAV particles in mice is missing. Therefore, we examined feces, urine, and saliva of mice after CNS injection with AAV2/8. It revealed the presence of viral DNA fragments via qPCR for up to 4 days after injection. To examine AAV functionality we performed nested PCR and could not detect full-length viral genomes in any but two collected feces samples. Furthermore, a functional infection assay did not reveal evidence of intact AAV particles. Our findings are supposed to contribute murine shedding data as a foundation to help establish still lacking adequate biosafety regulations in the context of AAV shedding.

Crimean-Congo Hemorrhagic Fever Virus Kinetics in Serum, Saliva, and Urine, Iran, 2018.

Little is known about using noninvasive samples for diagnosing Crimean-Congo hemorrhagic fever (CCHF). We investigated detection of CCHF virus in serum, saliva, and urine samples. Our results indicate that serum is the best sample type for CCHF diagnosis; saliva can be used for noninvasive sampling.

Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2.

The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses. IMPORTANCE: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.

Generation of recombinant viruses directly from clinical specimens of COVID-19 patients.

Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2-a good example of a practical target-and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the de novo generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens. IMPORTANCE: Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient in vitro replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases.

Detection and characterization of novel luchacoviruses, genus Alphacoronavirus, in saliva and feces of meso-carnivores in the northeastern United States.

IMPORTANCE: Several coronaviruses (CoVs) have been detected in domesticated, farmed, and wild meso-carnivores, causing a wide range of diseases and infecting diverse species, highlighting their important but understudied role in the epidemiology of these viruses. Assessing the viral diversity hosted in wildlife species is essential to understand their significance in the cross-species transmission of CoVs. Our focus here was on CoV discovery in meso-carnivores in the Northeast United States as a potential "hotspot" area with high density of humans and urban wildlife. This study identifies novel alphacoronaviruses circulating in multiple free-ranging wild and domestic species in this area and explores their potential epidemiological importance based on regions of the Spike gene, which are relevant for virus-host interactions.

Diagnostic performance, stability, and acceptability of self-collected saliva without additives for SARS-CoV-2 molecular diagnosis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19), leading to a global pandemic. The molecular diagnosis of this virus is mostly performed by collecting upper respiratory samples, which has many disadvantages, including patient discomfort and the need for trained healthcare professionals. Although saliva has emerged as a more comfortable sample, the use of additives to preserve viral RNA is expensive and, in some cases, difficult for self-collection. METHOD: This study evaluated the diagnostic performance by RT-PCR and stability of self-collected saliva using wide-mouth specimen collection cups without stabilization and/or inactivation buffers for SARS-CoV-2 detection, compared to nasopharyngeal samples and saliva collected with additives. Additionally, the study assessed the acceptability of this sample collection method among participants and healthcare personnel. RESULTS: The study included 1281 volunteers with a 24.6% positive infection rate. Saliva demonstrated comparable diagnostic performance to nasopharyngeal samples, with a sensitivity of 87.6% and specificity of 99.6%, for a total percent agreement of 96.4%. The study also showed that viral RNA in saliva remained stable for at least 72 h at different temperatures. Notably, saliva samples without additives exhibited a lower RdRp Ct compared to samples with additives, suggesting that the absence of stabilization and/or inactivation buffers does not significantly affect its performance. The study highlighted the acceptability of saliva among patients and healthcare personnel due to its noninvasive nature and ease of collection. CONCLUSIONS: This research supports the implementation of self-collected saliva as a comfortable and user-friendly alternative sample for SARS-CoV-2 diagnosis.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2.

BACKGROUND: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. METHODS: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. RESULTS: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen's kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity. CONCLUSIONS: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.

Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Oral mouthwashes for asymptomatic to mildly symptomatic adults with COVID-19 and salivary viral load: a randomized, placebo-controlled, open-label clinical trial.

BACKGROUND: Recent randomized clinical trials suggest that the effect of using cetylpyridinium chloride (CPC) mouthwashes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in COVID-19 patients has been inconsistent. Additionally, no clinical study has investigated the effectiveness of on-demand aqueous chlorine dioxide mouthwash against COVID-19. METHODS: We performed a randomized, placebo-controlled, open-label clinical trial to assess for any effects of using mouthwash on the salivary SARS-CoV-2 viral load among asymptomatic to mildly symptomatic adult COVID-19-positive patients. Patients were randomized to receive either 20 mL of 0.05% CPC, 10 mL of 0.01% on-demand aqueous chlorine dioxide, or 20 mL of placebo mouthwash (purified water) in a 1:1:1 ratio. The primary endpoint was the cycle threshold (Ct) values employed for SARS-CoV-2 salivary viral load estimation. We used linear mixed-effects models to assess for any effect of the mouthwashes on SARS-CoV-2 salivary viral load. RESULTS: Of a total of 96 eligible participants enrolled from November 7, 2022, to January 19, 2023, 90 were accepted for the primary analysis. The use of 0.05% CPC mouthwash was not shown to be superior to placebo in change from baseline salivary Ct value at 30 min (difference vs. placebo, 0.640; 95% confidence interval [CI], -1.425 to 2.706; P = 0.543); 2 h (difference vs. placebo, 1.158; 95% CI, -0.797 to 3.112; P = 0.246); 4 h (difference vs. placebo, 1.283; 95% CI, -0.719 to 3.285; P = 0.209); 10 h (difference vs. placebo, 0.304; 95% CI, -1.777 to 2.385; P = 0.775); or 24 h (difference vs. placebo, 0.782; 95% CI, -1.195 to 2.759; P = 0.438). The use of 0.01% on-demand aqueous chlorine dioxide mouthwash was also not shown to be superior to placebo in change from baseline salivary Ct value at 30 min (difference vs. placebo, 0.905; 95% CI, -1.079 to 2.888; P = 0.371); 2 h (difference vs. placebo, 0.709; 95% CI, -1.275 to 2.693; P = 0.483); 4 h (difference vs. placebo, 0.220; 95% CI, -1.787 to 2.226; P = 0.830); 10 h (difference vs. placebo, 0.198; 95% CI, -1.901 to 2.296; P = 0.854); or 24 h (difference vs. placebo, 0.784; 95% CI, -1.236 to 2.804; P = 0.447). CONCLUSIONS: In asymptomatic to mildly symptomatic adults with COVID-19, compared to placebo, the use of 0.05% CPC and 0.01% on-demand aqueous chlorine dioxide mouthwash did not lead to a significant reduction in SARS-CoV-2 salivary viral load. Future studies of the efficacy of CPC and on-demand aqueous chlorine dioxide mouthwash on the viral viability of SARS-CoV-2 should be conducted using different specimen types and in multiple populations and settings.

Oral human papillomavirus (HPV) infection and HPV vaccination in an Australian cohort.

Human papillomavirus (HPV)-related oropharyngeal cancer (OPC) is increasing in incidence, yet very little is known about oral HPV infection in the general population. In this Australian-based study we assess oral HPV prevalence according to HPV vaccination status. Participants of the Oral Diversity Study were Australian residents, aged 18 to 70 years, who filled out a questionnaire about lifestyle and sexual behaviour, and donated a saliva sample in 2020 to 2021. We obtained permission to access HPV vaccination status through record linkage with the Australian Immunisation Register. Saliva samples were DNA extracted, DNA quality checked and analysed for HPV. We recruited 1023 participants to the Oral Diversity Study. Nine hundred twenty-one returned a saliva sample for analysis, 911 passed the DNA quality check and were included in the study. The oral HPV prevalence was 7.2%, and was strongly associated with sexual behaviours. We identified 27 different HPV types; 53% of participants carried high-risk HPV types, with no difference between the vaccinated and the unvaccinated groups (53% both, P = .979). Two hundred thirty participants (26%) were HPV vaccinated. The oral prevalence of the nine HPV types included in the nonavalent HPV vaccine was significantly lower in the vaccinated participants compared to the unvaccinated (0.9% vs 3.4%; P = .022). These findings suggest that a sizeable minority of Australian residents harbour oral HPV infections, and many of these are high-risk subtypes. We found some evidence that HPV vaccination resulted in lower prevalence of oral HPV infections of vaccine-specific types. Larger surveys are required to confirm these findings.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

SARS-CoV-2 seroprevalence and determinants for salivary seropositivity among pupils and school staff: a prospective cohort study.

Representative school data on SARS-CoV-2 past-infection are scarce, and differences between pupils and staff remain ambiguous. We performed a nation-wide prospective seroprevalence study among pupils and staff over time and in relation to determinants of infection using Poisson regression and generalised estimating equations. A cluster random sample was selected with allocation by region and sociodemographic (SES) background. Surveys and saliva samples were collected in December 2020, March, and June 2021, and also in October and December 2021 for primary pupils. We recruited 885 primary and 569 secondary pupils and 799 staff in 84 schools. Cumulative seroprevalence (95% CI) among primary pupils increased from 11.0% (7.6; 15.9) at baseline to 60.4% (53.4; 68.3) in December 2021. Group estimates were similar at baseline; however, in June they were significantly higher among primary staff (38.9% (32.5; 46.4)) compared to pupils and secondary staff (24.2% (20.3; 28.8)). Infections were asymptomatic in 48-56% of pupils and 28% of staff. Seropositivity was associated with individual SES in pupils, and with school level, school SES and language network in staff in June. Associations with behavioural characteristics were inconsistent. Seroconversion rates increased two- to four-fold after self-reported high-risk contacts, especially with adults. Seroprevalence studies using non-invasive sampling can inform public health management.

The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission.

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant air environment, they disappear from the window of view with time constants in the range of 8 to 14 min, which corresponds to droplet nuclei of ca. 4 µm diameter, or 12- to 21-µm droplets prior to dehydration. These observations confirm that there is a substantial probability that normal speaking causes airborne virus transmission in confined environments.

Physics of virus transmission by speaking droplets.

To make the physics of person-to-person virus transmission from emitted droplets of oral fluid while speaking easily understood, we present simple and transparent algebraic equations that capture the essential physics of the problem. Calculations with these equations provide a straightforward way of determining whether emitted droplets remain airborne or rapidly fall to the ground, after accounting for the decrease in droplet size from water evaporation. At a relative humidity of 50%, for example, droplets with initial radii larger than about 50 µm rapidly fall to the ground, while smaller, potentially virus-containing droplets shrink in size from water evaporation and remain airborne for many minutes. Estimates of airborne virion emission rates while speaking strongly support the proposal that mouth coverings can help contain the COVID-19 pandemic.