Antigenic distance between primary and secondary dengue infections correlates with disease risk.

Many pathogens continuously change their protein structure in response to immune-driven selection, resulting in weakened protection even in previously exposed individuals. In addition, for some pathogens, such as dengue virus, poorly targeted immunity is associated with increased risk of severe disease through a mechanism known as antibody-dependent enhancement. However, it remains unclear whether the antigenic distances between an individual's first infection and subsequent exposures dictate disease risk, explaining the observed large-scale differences in dengue hospitalizations across years. Here, we develop a framework that combines detailed antigenic and genetic characterization of viruses with details on hospitalized cases from 21 years of dengue surveillance in Bangkok, Thailand, to identify the role of the antigenic profile of circulating viruses in determining disease risk. We found that the risk of hospitalization depended on both the specific order of infecting serotypes and the antigenic distance between an individual's primary and secondary infections, with risk maximized at intermediate antigenic distances. These findings suggest that immune imprinting helps determine dengue disease risk and provide a pathway to monitor the changing risk profile of populations and to quantifying risk profiles of candidate vaccines.

Molecular and Entomological Characterization of 2023 Dengue Outbreak in Dhading District, Central Nepal.

In 2023, Nepal faced its second largest dengue outbreak ever, following a record-breaking number of dengue cases in 2022, characterized by the expansion of infections into areas of higher altitudes. However, the characteristics of the 2023 circulating dengue virus (DENV) and the vector density remain poorly understood. Therefore, we performed DENV serotyping, clinical and laboratory assessment, and entomological analysis of the 2023 outbreak in central Nepal. A total of 396 fever cases in Dhading hospital suspected of being DENV positive were enrolled, and blood samples were collected and tested by different techniques including PCR. Of these, 278 (70.2%) had confirmed DENV infection. Multiple serotypes (DENV-1, -2, and -3) were detected. DENV-2 (97.5%) re-emerged after six years in Dhading while DENV-3 was identified for the first time. Dengue inpatients had significantly higher frequency of anorexia, myalgia, rash, diarrhea, nausea, vomiting, abdominal pain, and thrombocytopenia (p < 0.05). In this area, Aedes mosquitoes largely predominated (90.7%) with the majority being A. aegypti (60.7%). We also found high levels of Aedes index (20.0%) and container index (16.7%). We confirmed multiple DENV serotype circulation with serotype re-emergence and new serotype introduction, and high vector density in 2023. These findings call for the urgent initiation and scaling up of DENV molecular surveillance in human and mosquito populations for dengue control and prevention in Nepal.

An unusual diarrheal outbreak in the community in Eastern Thailand caused by Norovirus GII.3[P25].

BACKGROUND: Sentinel laboratory surveillance for diarrheal disease determined norovirus to be the most common cause of non-bacterial gastroenteritis in people during the COVID-19 pandemic in Thailand. An increase in patients presenting with diarrhea and vomiting in hospitals across Chanthaburi province between December 2021 and January 2022 led to the need for the identification of viral pathogens that may be responsible for the outbreak. METHODS: Fecal samples (rectal swabs or stool) from 93 patients, of which 65 patients were collected during the December 2021 to January 2022 outbreak, were collected and screened for viral infection by real-time RT-PCR. Positive samples for norovirus GII were then genotyped by targeted amplification and sequencing of partial polymerase and capsid genes. Full genome sequencing was performed from the predominant strain, GII.3[P25]. RESULTS: Norovirus was the most common virus detected in human fecal samples in this study. 39 of 65 outbreak samples (60%) and 3 of 28 (10%) non-outbreak samples were positive for norovirus genogroup II. One was positive for rotavirus, and one indicated co-infection with rotavirus and norovirus genogroups I and II. Nucleotide sequences of VP1 and RdRp gene were successfully obtained from 28 of 39 positive norovirus GII and used for dual-typing; 25/28 (89.3%) were GII.3, and 24/28 (85.7) were GII.P25, respectively. Norovirus GII.3[P25] was the predominant strain responsible for this outbreak. The full genome sequence of norovirus GII.3[P25] from our study is the first reported in Thailand and has 98.62% and 98.57% similarity to norovirus found in China in 2021 and the USA in 2022, respectively. We further demonstrate the presence of multiple co-circulating norovirus genotypes, including GII.21[P21], GII.17[P17], GII.3[P12] and GII.4[P31] in our study. CONCLUSIONS: An unusual diarrhea outbreak was found in December 2021 in eastern Thailand. Norovirus strain GII.3[P25] was the cause of the outbreak and was first detected in Thailand. The positive rate during GII.3[P25] outbreak was six times higher than sporadic cases (GII.4), and, atypically, adults were the primary infected population rather than children.

Respiratory syncytial virus among hospitalized patients of severe acute respiratory infection in Bhutan: Cross-sectional study.

Introduction: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, particularly in young children. In Bhutan, respiratory disease continues to be among the top 10 diseases of morbidity for several years. This study aimed to estimate the prevalence of RSV among hospitalized patients with severe acute respiratory infection (SARI) in Bhutan. Method: Respiratory specimens were collected from SARI patients of all ages in 2016 and 2018 following influenza surveillance guidelines. Specimens were tested for influenza and RSV, human metapneumovirus, adenovirus, and human parainfluenza virus types 1, 2, and 3 using real-time reverse-transcription polymerase chain reaction assay. Descriptive statistics were used to analyze the result in STATA 16.1. Result: Of the 1339 SARI specimens tested, 34.8% were positive for at least one viral pathogen. RSV was detected in 18.5% of SARI cases, followed by influenza in 13.4% and other respiratory viruses in 3%. The median age of SARI cases was 3 (IQR: 0.8-21 years) years. RSV detection was higher among children aged 0-6 (Adj OR: 3.03; 95% CI: 1.7-5.39) and 7-23 months (Adj OR: 3.01; 95% CI: 1.77-5.12) compared with the children aged 5-15 years. RSV was also associated with breathing difficulty (Adj OR: 1.73; 95% CI: 1.17-2.56) and pre-existing lung disease, including asthma (Adj OR: 2.78; 95% CI: 0.99-7.8). Conclusion: Respiratory viruses were detected in a substantial proportion of SARI hospitalizations in Bhutan.

High Transmission Rates of Early Omicron Subvariant BA.2 in Bangkok, Thailand.

The emergence of Omicron as the fifth variant of concern within the SARS-CoV-2 pandemic in late 2021, characterized by its rapid transmission and distinct spike gene mutations, underscored the pressing need for cost-effective and efficient methods to detect viral variants, especially given their evolving nature. This study sought to address this need by assessing the effectiveness of two SARS-CoV-2 variant classification platforms based on RT-PCR and mass spectrometry. The primary aim was to differentiate between Delta, Omicron BA.1, and Omicron BA.2 variants using 618 COVID-19-positive samples collected from Bangkok patients between November 2011 and March 2022. The analysis revealed that both BA.1 and BA.2 variants exhibited significantly higher transmission rates, up to 2-3 times, when compared to the Delta variant. This research presents a cost-efficient approach to virus surveillance, enabling a quantitative evaluation of variant-specific public health implications, crucial for informing and adapting public health strategies.

Antigenic diversity and dengue disease risk.

Many pathogens continuously change their protein structure in response to immune-driven selection, resulting in weakened protection. In addition, for some pathogens such as dengue virus, poorly targeted immunity is associated with increased risk of severe disease, through a mechanism known as antibody-dependent enhancement. However, it remains a mystery whether the antigenic distance between an individual's first infection and subsequent exposures dictate disease risk, explaining the observed large-scale differences in dengue hospitalisations across years. Here we develop an inferential framework that combines detailed antigenic and genetic characterisation of viruses, and hospitalised cases from 21 years of surveillance in Bangkok, Thailand to identify the role of the antigenic profile of circulating viruses in determining disease risk. We find that the risk of hospitalisation depends on both the specific order of infecting serotypes and the antigenic distance between an individual's primary and secondary infections, with risk maximised at intermediate antigenic distances. These findings suggest immune imprinting helps determine dengue disease risk, and provides a pathway to monitor the changing risk profile of populations and to quantifying risk profiles of candidate vaccines.

Secondary dengue serotype 1 infection causing dengue shock syndrome with rhombencephalitis and bleeding associated with refractory thrombocytopenia: A case report.

Background: Dengue has a wide spectrum of manifestations, from an asymptomatic condition to dengue shock syndrome. Extensive plasma leakage, severe bleeding, or both, could lead to dengue shock syndrome, a common cause of death in dengue-infected patients. Thrombocytopenia is a common laboratory finding in dengue, which correlates with the disease severity and rapidly resolves during the recovery phase. Therefore, refractory thrombocytopenia is rare in patients with dengue. Rhombencephalitis is an inflammatory disease affecting the hindbrain, rarely associated with dengue. We report the second case of dengue-associated rhombencephalitis, wherein the patient developed dengue shock syndrome and severe bleeding associated with refractory thrombocytopenia. Case report: A 47-year-old Thai female with secondary dengue serotype 1 infection developed dengue shock syndrome with rhombencephalitis, manifested as altered sensorium and status epilepticus in the critical phase. Cerebrospinal fluid analysis showed pleocytosis with predominantly mononuclear cells and high protein levels. Magnetic resonance imaging of the brain showed multifocal brain signal abnormalities involving the medulla oblongata, pons, midbrain, bilateral hippocampi, thalami, posterior limb of internal capsules, external capsules, and deep hemispheric white matter. The patient had partial neurological recovery following rhombencephalitis for one month. During the recovery phase, severe bleeding with refractory thrombocytopenia and acute kidney injury were observed. Methylprednisolone with eltrombopag was administered, which resulted in an increased the platelet count, cessation of bleeding and recovery of kidney function within 4 days. Conclusions: Dengue is a potential cause of rhombencephalitis. Dengue-associated rhombencephalitis develops during the critical phase, with only partial neurological recovery. However, severe bleeding and refractory thrombocytopenia were also observed during the recovery phase. Methylprednisolone with a thrombopoietin receptor agonist could be an effective treatment for increasing platelet count and stopping bleeding in dengue.

SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region.

Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for comparison to standard clinical sampling. A total of 215 wastewater samples were collected and tested for SARS-CoV-2 RNA by real-time PCR with three targeted genes (N, E, and ORF1ab); 102 samples were positive (42.5%). The SARS-CoV-2 variants were determined by a multiplex PCR MassARRAY assay to distinguish four SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Multiple variants of Alpha-Delta and Delta-Omicron were detected in the wastewater samples in July 2021 and January 2022, respectively. These wastewater variant results mirrored the country data from clinical specimens deposited in GISAID. Our results demonstrated that wastewater surveillance using multiple signature mutation sites for SARS-CoV-2 variant detection is an appropriate strategy to monitor the presence of SARS-CoV-2 variants in the community at a low cost and with rapid turn-around time. However, it is essential to note that sequencing surveillance of wastewater samples should be considered complementary to whole genome sequencing of clinical samples to detect novel variants.

Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Influenza-Like Symptom Incidence, Illness-Associated Expenses, and Economic Impact Among Healthcare Personnel in Thailand: A Prospective Observational Cohort Study (2020-2021).

OBJECTIVES: We estimated influenza-like symptom (ILS) incidence among healthcare personnel (HCP) in four hospitals and the economic impact due to ILS in the Thai HCP population during July 2020-June 2021 (Thailand's expected 2020 influenza season), which also coincided with the novel coronavirus disease 2019 pandemic. METHODS: We followed HCP, in a prospective observational cohort, weekly for ≥1 of: muscle pain, cough, runny nose/nasal congestion, sore throat, or difficulty breathing. We fitted population-averaged Poisson regression models to identify factors associated with acquiring ILS and to calculate ILS incidence. We applied epidemiologic parameters to Thailand's HCP population (227 349 persons) to estimate economic impact. RESULTS: Of 2184 participants, adjusted all-cause ILS incidence was 6.1 episodes per 100 person-years (95% confidence interval 3.4-10.9). Among Thailand's HCP population, 13 909 ILS episodes were estimated to occur annually and would result in US$235 135 economic loss. Controlling for study site and calendar month, factors associated with acquiring ≥1 ILS versus no ILS included being female, having asthma, and using personal protective equipment 'frequently, but not always'. CONCLUSIONS: All-cause ILS resulted in considerable economic loss among Thai HCP workforce. These findings underscore the importance of public health interventions to reduce the risk of acquiring ILS.

Anti-Arbovirus Antibodies Cross-React With Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is found in regions where dengue (DENV) and chikungunya (CHIKV) viruses are endemic. Any serological cross-reactivity between DENV, CHIKV, and SARS-CoV-2 is significant as it could lead to misdiagnosis, increased severity, or cross-protection. This study examined the potential cross-reactivity of anti-DENV and CHIKV antibodies with SARS-CoV-2 using acute and convalescent-phase samples collected before the SARS-CoV-2 pandemic. These included healthy, normal human (NHS, n = 6), CHIKV-positive (n = 14 pairs acute and convalescent), primary DENV-positive (n = 20 pairs), secondary DENV-positive (n = 20 pairs), and other febrile illnesses sera (n = 23 pairs). Samples were tested using an in-house SARS-CoV-2 and a EUROIMMUN IgA and IgG ELISAs. All NHS samples were negative, whereas 3.6% CHIKV, 21.7% primary DENV, 15.7% secondary DENV, and 10.8% febrile diseases sera resulted as anti-SARS-CoV-2 antibody positive. The EUROIMMUN ELISA using spike 1 as the antigen detected more positives among the primary DENV infections than the in-house ELISA using spike 1-receptor binding domain (RBD) protein. Among ELISA-positive samples, four had detectable neutralizing antibodies against SARS-CoV-2 reporter virus particles yet none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated the SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. IMPORTANCE SARS-CoV-2 continues to cause significant morbidity globally, including in areas where DENV and CHIKV are endemic. Reports using rapid diagnostic and ELISAs have demonstrated that serological cross-reactivity between DENV and SARS-CoV-2 can occur. Furthermore, it has been observed that convalescent DENV patients are at a lower risk of developing COVID-19. This phenomenon can interfere with the accuracy of serological testing and clinical management of both DENV and COVID-19 patients. In this study, the cross-reactivity of primary/secondary anti-DENV, CHIKV, and other febrile illness antibodies with SARS-CoV-2 using two ELISAs has been shown. Among ELISA-positive samples, four had detectable levels of neutralizing antibodies against SARS-CoV-2 reporter virus particles. However, none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. The data discussed here provide information regarding diagnosis and may help guide appropriate public health interventions.

Homologous or Heterologous COVID-19 Booster Regimens Significantly Impact Sero-Neutralization of SARS-CoV-2 Virus and Its Variants.

We determined the levels of neutralizing antibodies against the SARS-CoV-2 ancestral strain, Delta and Omicron variants of concern (VOCs), in 125 healthcare workers who received CoronaVac as their primary vaccination and later received either a single ChAdOx1 or a combi-nation of two consecutive boosters using either two ChAdOx1 doses or a ChAdOx1 or BNT162b2 as the primary and second boosters, respectively, or two doses of BNT162b2. The titers 12 weeks after primary vaccination were inadequate to neutralize all strains. After a single ChAdOx1 booster, the levels of neutralization at Day 30 varied significantly, with only a small proportion of participants developing neutralizing titers against Omicron at Day 7 and 30. The two doses of ChAdOx1 as the booster induced the lowest activity. A combination ChAdOx1 and BNT162b2 induced greater neutralization than by two doses of ChAdOx1. Two doses of BNT162b2 as the booster had the maximal activity against Omicron VOC.

Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: Protective efficacy, immunogenicity, safety, and toxicity.

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.

Clinical evaluation of the BioFire Global Fever Panel for the identification of malaria, leptospirosis, chikungunya, and dengue from whole blood: a prospective, multicentre, cross-sectional diagnostic accuracy study.

BACKGROUND: Acute febrile illness is a common presentation for patients at hospitals globally. Assays that can diagnose a variety of common pathogens in blood could help to establish a diagnosis for targeted disease management. We aimed to evaluate the performance of the BioFire Global Fever Panel (GF Panel), a multiplex nucleic acid amplification test performed on whole blood specimens run on the BioFire FilmArray System, in the diagnosis of several pathogens that cause acute febrile illness. METHODS: We did a prospective, multicentre, cross-sectional diagnostic accuracy study to evaluate the GF Panel. Consenting adults and children older than 6 months presenting with fever in the previous 2 days were enrolled consecutively in sub-Saharan Africa (Ghana, Kenya, Tanzania, Uganda), southeast Asia (Cambodia, Thailand), central and South America (Honduras, Peru), and the USA (Washington, DC; St Louis, MO). We assessed the performance of six analytes (chikungunya virus, dengue virus [serotypes 1-4], Leptospira spp, Plasmodium spp, Plasmodium falciparum, and Plasmodium vivax or Plasmodium ovale) on the GF Panel. The performance of the GF Panel was assessed using comparator PCR assays with different primers followed by bidirectional sequencing on nucleic acid extracts from the same specimen. We calculated the positive percent agreement and negative percent agreement of the GF Panel with respect to the comparator assays. This study is registered with ClinicalTrials.gov, NCT02968355. FINDINGS: From March 26, 2018, to Sept 30, 2019, 1965 participants were enrolled at ten sites worldwide. Of the 1875 participants with analysable results, 980 (52·3%) were female and the median age was 22 years (range 0-100). At least one analyte was detected in 657 (35·0%) of 1875 specimens. The GF Panel had a positive percent agreement for the six analytes evaluated as follows: chikungunya virus 100% (95% CI 86·3-100), dengue virus 94·0% (90·6-96·5), Leptospira spp 93·8% (69·8-99·8), Plasmodium spp 98·3% (96·3-99·4), P falciparum 92·7% (88·8-95·6), and P vivax or P ovale 92·7% (86·7-96·6). The GF Panel had a negative percent agreement equal to or greater than 99·2% (98·6-99·6) for all analytes. INTERPRETATION: This 1 h sample-to-answer, molecular device can detect common causative agents of acute febrile illness with excellent positive percent agreement and negative percent agreement directly in whole blood. The targets of the assay are prevalent in tropical and subtropical regions globally, and the assay could help to provide both public health surveillance and individual diagnoses. FUNDING: BioFire Defense, Joint Project Manager for Medical Countermeasure Systems and US Army Medical Materiel Development Activity, and National Institute of Allergy and Infectious Diseases.

Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand.

Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok, Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within ±3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances compared to E alone (50.5% to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (ΔRMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface.

Assessing the role of multiple mechanisms increasing the age of dengue cases in Thailand.

The mean age of dengue hemorrhagic fever (DHF) cases increased considerably in Thailand from 8.1 to 24.3 y between 1981 and 2017 (mean annual increase of 0.45 y). Alternative proposed explanations for this trend, such as changes in surveillance practices, reduced mosquito­human contact, and shifts in population demographics, have different implications for global dengue epidemiology. To evaluate the contribution of each of these hypothesized mechanisms to the observed data, we developed 20 nested epidemiological models of dengue virus infection, allowing for variation over time in population demographics, infection hazards, and reporting rates. We also quantified the effect of removing or retaining each source of variation in simulations of the age trajectory. Shifts in the age structure of susceptibility explained 58% of the observed change in age. Adding heterogeneous reporting by age and reductions in per-serotype infection hazard to models with shifts in susceptibility explained an additional 42%. Reductions in infection hazards were mostly driven by changes in the number of infectious individuals at any time (another consequence of shifting age demographics) rather than changes in the transmissibility of individual infections. We conclude that the demographic transition drives the overwhelming majority of the observed change as it changes both the age structure of susceptibility and the number of infectious individuals. With the projected Thai population age structure, our results suggest a continuing increase in age of DHF cases, shifting the burden toward individuals with more comorbidity. These insights into dengue epidemiology may be relevant to many regions of the globe currently undergoing comparable changes in population demographics.

Chimeric Virus-like Particle-Based COVID-19 Vaccine Confers Strong Protection against SARS-CoV-2 Viremia in K18-hACE2 Mice.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

Clinical characterization of COVID-19 breakthrough infections, Philippines.

We clinically characterized PCR detected breakthrough infections among partially/fully vaccinated cases with majority given an inactivated vaccine, CoronaVac. From 1 March to 15 July 2021, we detected 182 SARS-CoV-2 infections among vaccinated cases with 129 classified as breakthrough infections. Majority were male, 30-39 y.o., and were asymptomatic or mildly symptomatic with few severe cases. Alpha, Beta and Delta VOCs were detected from sequenced breakthrough infections. Healthcare workers had significantly lower Ct values(higher viral loads) versus non-HCWs. Our results underscore the importance of regular PCR screening for HCWs due to the risk of SARS-CoV-2 transmission from asymptomatic breakthrough infections and provide evidence supporting administration of a booster dose especially to HCWs.

Neurocognitive impact of Zika virus infection in adult rhesus macaques.

BACKGROUND: Zika virus (ZIKV) is a mosquito-transmitted flavivirus that affects many regions of the world. Infection, in utero, causes microcephaly and later developmental and neurologic impairments. The impact of ZIKV infection on neurocognition in adults has not been well described. The objective of the study was to assess the neurocognitive impact of ZIKV infection in adult rhesus macaques. METHODS: Neurocognitive assessments were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) via a touch screen and modified Brinkman Board before and after subcutaneous ZIKV inoculation. Immune activation markers were measured in the blood and cerebral spinal fluid (CSF) by multiplex assay and flow cytometry. RESULTS: All animals (N = 8) had detectable ZIKV RNA in plasma at day 1 post-inoculation (PI) that peaked at day 2 PI (median 5.9, IQR 5.6-6.2 log10 genome equivalents/mL). In all eight animals, ZIKV RNA became undetectable in plasma by day 14 PI, but persisted in lymphoid tissues. ZIKV RNA was not detected in the CSF supernatant at days 4, 8, 14 and 28 PI but was detected in the brain of 2 animals at days 8 and 28 PI. Elevations in markers of immune activation in the blood and CSF were accompanied by a reduction in accuracy and reaction speed on the CANTAB in the majority of animals. CONCLUSIONS: The co-occurrence of systemic and CSF immune perturbations and neurocognitive impairment establishes this model as useful for studying the impact of neuroinflammation on neurobehavior in rhesus macaques, as it pertains to ZIKV infection and potentially other pathogens.