Neutralization Test Research Articles

Recombinant receptor-binding motif of spike COVID-19 vaccine candidate induces SARS-CoV-2 neutralizing antibody response

Introduction: The SARS-CoV-2 pandemic necessitates effective therapeutic solutions. The receptor-binding motif (RBM) is a subdomain of the spike protein's receptor-binding domain (RBD) and is critical for facilitating the binding of SARS-CoV-2 to the human ACE2 receptor. This study investigates the use of the receptor-binding motif (RBM) domain as an immunogen to produce potent neutralizing antibodies against SARS-CoV-2. Methods: The RBM gene was codon-optimized and cloned into the pET17b vector for expression in E. coli BL21 (DE3) cells, induced with 1 mM IPTG. The recombinant RBM protein was purified using Ni-NTA affinity chromatography. After validating the recombinant RBM by Western blotting with anti-His tag antibodies, BALB/c mice were immunized with 20 µg of the purified RBM protein. Anti-RBM IgG was subsequently purified using protein G resin, and its neutralizing capacity was assessed using the Pishtaz Teb Zaman Neutralization Assay Kit. Results: The recombinant RBM protein, with a molecular weight of 10 kDa, was expressed as inclusion bodies. the typical yield of purification was 27 mg/L of bacterial culture. The neutralization test demonstrated a concentration of 36 µg/mL of neutralizing antibodies in the immunized serum, preventing the spike protein from binding to ACE2. Conclusion: Our study demonstrated that anti-RBM antibodies exhibited neutralization effects on SARS-CoV-2. These findings provide evidence for the development of a vaccine candidate through the induction of antibodies against the RBM, necessitating further studies with adjuvants suitable for human use to evaluate its potential for human vaccination.

A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge. The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine. The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

Long-term dynamics of measles virus-specific neutralizing antibodies in children vaccinated before 12 months of age.

Measles is a highly contagious disease presenting a significant risk for unvaccinated infants and adults. Measles vaccination under the age of 12 months provides early protection, but has also been associated with blunting of antibody responses to subsequent measles vaccinations and assumed to have lower vaccine effectiveness. Our study included children who received an early measles, mumps and rubella (MMR) vaccination between 6 and 12 months of age (n=79, given in addition to the regular MMR vaccination schedule at 14 months and 9 years) and a group without additional early vaccination (n=44). We evaluated measles virus (MeV)-specific neutralizing antibodies before vaccination at 14 months and up to 6 years thereafter using a plaque reduction neutralization test according to the standard set by the WHO. We show a significant association between age of first MMR and MeV-specific neutralizing antibody levels later in life. Although most children who received early vaccination seroconverted after the first dose, children vaccinated before 8·5 months of age exhibited a markedly faster antibody decay and lost their protective neutralizing antibody levels over 6 years. Routine vaccination of infants under 8·5 months of age may lead to blunted MeV-specific antibody responses to subsequent MMR vaccination. Early MMR vaccination should only be considered during measles outbreaks or in other situations of increased risk of MeV infection.

Integrated Genetic and Statolith Shape Analysis Reveals the Population Structure of Loliolus (Nipponololigo) uyii (Cephalopoda: Loliginidae) in the Coastal Waters of China

Understanding population structure is a priority for evaluating population dynamics of commercially fished cephalopods under fishing pressure and environmental changes. This study employed a multidisciplinary approach to clarify the population structure of Loliolus (Nipponololigo) uyii, a common squid in inshore fisheries. Sampling was conducted multiple times to cover the distribution range across the East China Sea and South China Sea. High haplotype diversity was revealed by three gene markers (COI, 16S and ODH). Two geographical clades with significant genetic differentiation were divided through phylogenetic trees and haplotype networks. The boundary between the two clades is delineated by the Dongshan population in the southern East China Sea. Furthermore, the neutrality tests and mismatch analysis suggested that L. (N.) uyii populations may have undergone population expansion. Correspondingly, statolith differences in lateral dome and posterior indentation, along with high classification success, further supported the genetic division. The overall difference in statolith shape also efficiently identified seasonal groups in the Beibu Gulf lacking genetic differentiation. This result offers new insights into the influence of genetic and environmental factors on statolith shape. The integrated results provide a comprehensive understanding of the population structure of L. (N.) uyii, laying the foundation for resource development and the conservation of the species.

Genetic diversity of Plasmodium vivax population in northeast Myanmar assessed by amplicon sequencing of PvMSP1 and PvMSP3α

This study aimed to assess the baseline genetic diversity of the Plasmodium vivax population in an endemic area of northeast Myanmar at the onset of the malaria elimination campaign in the Greater Mekong Subregion. We genotyped 125 P. vivax clinical samples at two merozoite surface protein loci, PvMSP1 and PvMSP3α, by amplicon deep sequencing. Our study revealed that the parasite population in this region was highly diverse, identifying 60 PvMSP1 and 98 PvMSP3α haplotypes, with haplotype diversity of 0.929 and 0.944, respectively. Remarkably, 97.6% (122/125) of the patients harbored multiclonal infections with a mean multiplicity of infection of 4.18, indicating a relatively high transmission intensity. Neutrality tests and network analysis suggested a recent parasite population expansion, consistent with the concurrent malaria outbreak in the region. These findings underscore the existence of a highly diverse P. vivax population at the China-Myanmar border, highlighting the need for effective malaria control strategies to achieve the goal of regional malaria elimination.

Morphological analysis and cytotoxicity of acrylamide on SPC212 human mesothelioma cells: Do low doses induce proliferation, while high doses cause toxicity?

Acrylamide is broadly utilized in numerous areas with different purposes including being an additive, flocculating, sealing, dry strength improver and polymerizing agent, and so forth. Furthermore, it forms in certain food products at high temperatures. It poses serious hazard since its readily water-soluble and very reactive nature. Besides invivo studies, several invitro studies with various cell lines are carried out to evaluate its toxicity. However, of these cell line studies, there are no mesothelium or mesothelioma cell lines. To fill this lacuna, we aimed at examining various dose range of acrylamide on SPC212 human mesothelioma cell line. First, we executed MTT and neutral red cytotoxicity tests and ascertained IC50 dose. Next, we performed inverted, light (haematoxylin-eosin and May Grünwald), fluorescent (DAPI) and confocal microscope (AO/EB) analyses as well as immunohistochemistry for Bax, Bcl-2 and PCNA proteins. As a result, we found IC50 of acrylamide at 2.65 mM. Starting from 3.13 mM of acrylamide dose, a deep decrease in cell proliferation was observed. Particularly in MTT assay, a proliferative action of acrylamide was detected at 0.39 and 0.78 mM, supported with inverted microscope images. In light microscope analysis, several cellular degenerations, including condensed and kidney-shaped nucleus were evident. In AO/EB staining, cells with apoptotic characteristics augmented dose-dependently, being upheld by a parallel uptick in Bax and a dimunition in Bcl-2 staining. Besides, PCNA decreased at IC50 dose of acrylamide. This is the acrylamide-associated first study conducted on SPC212 mesothelioma cells encompassing advanced morphological analysis. We believe this study to be an incentive for future studies.

Development of Human Monoclonal Antibodies With Broad Reactivity for Rabies Postexposure Prophylaxis.

Rabies is an acute lethal disease causing by the neurotropic virus rabies virus (RABV). Rabies immune globulin (RIG) as an indispensable component of rabies postexposure prophylaxis (PEP) always faces with great challenges in terms of costs, stability and safety. Our objective is to develop a novel and potential fully human monoclonal antibodies (mAbs) cocktail for the improvement of rabies PEP. The neutralizing fully human mAbs were screened by using fully humanized antibody mice (CAMouseHG). Then, two mAbs 26-12 G and 5-7 G were selected with potential neutralizing activity to RABV by using fluorescent antibody virus neutralization test (FAVN), which specifically bind to antigenic sites I and III of RABV-glycoprotein (RABV-G), the key amino acid residues were further identified in position 336, 337 of 5-7 G and 226, 227, 228 of 26-12 G by using cross-linking and mass-spectrometry. Both mAbs are highly conserved across 8 RABV strains (distributing in 3 lineages: Asian, Cosmopolitan and Arctic-related) and 1 IRKV strain, and showed high neutralizing potential. Moreover, the in vivo experiment demonstrated that our cocktail can protect Kunming mice from a lethal RABV challenge. Collectively, we generate two noncompeting fully human mAbs (26-12 G, 5-7 G) and obtained cocktail CAM001 with their mixture. The high-potency and broad-spectrum neutralization of the cocktail supports its utility in human rabies PEP as an efficacious and affordable alternative to RIG products, particularly in endemic areas.

Cattle, sheep, and goat humoral immune responses against SARS-CoV-2

Following the emergence of SARS-CoV-2 in late 2019, several species of domestic and wild animals have been found to be susceptible to SARS-CoV-2 infection through experimental inoculation and animal surveillance activities. Detection of SARS-CoV-2 specific antibodies in animals is an important surveillance tool since viral shedding in animals can only be detected for a short period of time. In this study, convenience serum samples were collected from 691 cattle, 698 sheep, and 707 goats from several regions in the United States, between 2019 to 2022. The samples were evaluated for the presence of SARS-CoV-2 specific antibodies using two commercial enzyme-linked immunosorbent assays (ELISA); one based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) and the other based on the nucleocapsid protein (N-ELISA) of SARS-CoV-2. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT) employing the Wuhan-like SARS-CoV-2 USA/WA1/2020 isolate. Our results indicate that ∼1% (6/691) of cattle, ∼2% (13/698) of sheep, and ∼2.5% (18/707) of goat serum samples were positive when using the sVNT, whereas ∼4% of cattle (25/691) and sheep (27/698), and 2.5% (18/707) of goat serum samples tested positive with the N-ELISA. None of the sVNT positive cattle, sheep, or goat serum samples had detectable neutralizing antibody activity (<1:8) against the SARS-CoV-2 USA/WA1/2020 isolate by the VNT. Our results indicate low seropositivity in cattle, sheep, and goats in the U.S., indicating the importance to continue monitoring for SARS-CoV-2 prevalence in animal species that are in close contact with humans.

Particle dynamics and QPOs around a black hole coupled with nonlinear electrodynamics and cloud of strings

We explore the motion of particles around a black hole coupled with nonlinear electrodynamics and a cloud of strings and examine the effects of the parameters of the black hole on the motion of particles. This black hole spacetime consists of five parameters, M the mass of the black hole, α the deviation parameters that determine the deviation from the Bardeen black hole, Q the magnetic charge, β which describes the cloud of strings, and the parameter K which describes the cosmological constant. We investigate the effect of these involved parameters on different physical quantities like horizon structure, effective potential, specific energy, angular momentum, innermost stable circular orbit and trajectories. Effective force is also discussed within the scope of different values of these involved parameters. We explore the oscillatory motion of neutral particles by perturbing the equations of motion near the stable circular orbits. Frequencies measured by local and distant observers are also calculated for the different choices of the involved parameters. Interestingly, Periastron precession is also investigated for the considered black hole solution. Lastly, we analyze the collision of two neutral test particles. All the investigated results are summarized with physically required behavior.

Cilgavimab and tixagevimab as pre-exposure prophylaxis in vaccine non-responder kidney transplant recipients during a period of prevalent SARS-CoV-2 BA.2 and BA.4/5 variants—a prospective cohort study (RESCUE-TX)

The response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination is severely impaired in patients on maintenance immunosuppression after kidney transplantation. We conducted a prospective cohort study of 194 kidney transplant recipients (KTR) who exhibited no response to SARS-CoV-2 vaccinations (i.e., SARS-CoV-2 spike protein antibodies ≤264 U/mL) and had no prior documented infection. Patients received 300mg of cilgavimab/tixagevimab as SARS-CoV-2 pre-exposure prophylaxis (PrEP) between March 4, 2022, and May 3, 2022 and were contrasted to a matched cohort of 186 KTRs also without immunization again defined as SARS-CoV-2 spike protein antibodies ≤264 U/mL and no documented prior infection. The primary outcome was the serum kinetics of cilgavimab/tixagevimab, the secondary endpoints were time to SARS-CoV-2 breakthrough infection, severity of disease and variant specific live viral invitro neutralization tests of patient sera. Longitudinal serum level monitoring showed a half-life of 91 days for both antibodies (95% CI 86-95 days for cilgavimab and 85-96 days for tixagevimab) in KTRs. Invitro neutralization tests showed effectiveness against the BA.2 omicron subvariant but not BA.5. The cumulative incidence of SARS-CoV-2 infections until May 15, 2022, (BA.2 dominance) was 15/194 vs 36/186 in the PrEP and control group respectively (OR=0.35, 95% CI 0.18-0.66) but was not different thereafter (BA.4/5 dominance). The number of severe infections during the BA.2 period was lower in the prophylaxis than in the control group (OR=0.37, 95% CI 0.17-0.79). This study showed that SARS-CoV-2 PrEP with cilgavimab/tixagevimab demonstrated clinical effectiveness against variants that are neutralised (BA.2) but not against BA.4/5. This study was funded by the Medical University of Vienna and an unrestricted grant from AstraZeneca (ESR-21-21585).

Measures of Population Immunity Can Predict the Dominant Clade of Influenza A (H3N2) in the 2017-2018 Season and Reveal Age-Associated Differences in Susceptibility and Antibody-Binding Specificity.

For antigenically variable pathogens such as influenza, strain fitness is partly determined by the relative availability of hosts susceptible to infection with that strain compared with others. Antibodies to the hemagglutinin (HA) and neuraminidase (NA) confer substantial protection against influenza infection. We asked if a cross-sectional antibody-derived estimate of population susceptibility to different clades of influenza A (H3N2) could predict the success of clades in the following season. We collected sera from 483 healthy individuals aged 1 to 90 years in the summer of 2017 and analyzed neutralizing responses to the HA and NA of representative strains using focus reduction neutralization tests (FNRT) and enzyme-linked lectin assays (ELLA). We estimated relative population-average and age-specific susceptibilities to circulating viral clades and compared those estimates to changes in clade frequencies in the following 2017-2018 season. The clade to which neutralizing antibody titers were lowest, indicating greater population susceptibility, dominated the next season. Titer correlations between viral strains varied by age, suggesting age-associated differences in epitope targeting driven by shared past exposures. Yet substantial unexplained variation remains within age groups. This study indicates how representative measures of population immunity might improve evolutionary forecasts and inform selective pressures on influenza.

The Re-Emergence of Neuroinvasive Flaviviruses in Croatia During the 2022 Transmission Season.

(Re-)emerging arboviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Usutu virus (USUV), are continuously increasing in incidence. We analyzed the epidemiological characteristics of flavivirus infections in humans, sentinel animals, and mosquitoes detected in the 2022 transmission season in Croatia. From April to November 2022, 110 hospitalized patients with neuroinvasive diseases (NID) were tested for the presence of arboviruses. RT-qPCR was used to detect TBEV, WNV, and USUV RNA. An ELISA and virus neutralization tests were used for the detection of flavivirus antibodies. TBEV infection was confirmed in 22 patients with NID. WNV NID was detected in six patients. TBE showed male predominance (81.8%; male-to-female ratio of 4.5:1). All but one WNV patients were males. TBE occurred from April to August, with the majority of patients (83.3%) being detected during the May-June-July period. WNV infections were recorded in August and September. In addition to human cases, asymptomatic WNV infections (IgM positive) were reported in 10 horses. For the first time in Croatia, WNV NID was observed in one horse that presented with neurological symptoms. Furthermore, USUV was confirmed in one dead blackbird that presented with neurological symptoms. A total of 1984 mosquitoes were collected in the City of Zagreb. Two Ae. albopictus pools tested positive for flavivirus RNA: one collected in July (USUV) and the other collected in August (WNV). A phylogenetic analysis of detected human and avian strains confirmed WNV lineage 2 and the USUV Europe 2 lineage. The presented results confirm the endemic presence of neuroinvasive flaviviruses in continental Croatia. The continuous monitoring of virus circulation in humans, sentinel animals, and mosquitoes is needed to reduce the disease burden.

Prediction, modeling, and optimization studies of eco-friendly gold extraction using alpha-cyclodextrin and the RSM and CCD methods

In the Democratic Republic of the Congo, gold extraction from ore in South Kivu utilized alpha-cyclodextrin (α-CD) as an eco-friendly method to replace harmful methods. Analysis of variance and response surface methodology were employed for prediction, modeling, and optimization studies. Atomic absorption spectroscopy revealed a 0.06% gold content in a randomly taken ore sample. Leaching with modified aqua regia was optimized with the following parameters: 7.27 h, 50 g/L HBr concentration, pH 1, and 200 rpm stirring speed, resulting in 98.5% removal. Neutralization tests with potassium hydroxide (KOH) followed leaching to prepare the solution medium with appropriate pH for extraction test, by varying parameters such as time, KOH concentration, and pH. Following neutralization, extraction tests with α-CD were carried out and optimized with the following parameters: 40 min, α-CD concentration of 11.6166 g/L, and a pH of 5, resulting in a percent removal of 98.9%. α-CD’s eco-friendly nature, cost-effectiveness, and high percent removal make it a promising and sustainable alternative for gold recovery, appealing to the mining industry.

Correlation Between the Anticorrosive Performance of Protective Coatings Under Neutral Salt Spray Testing and Outdoor Atmospheric and Immersion Exposure

Anticorrosive organic coatings are usually tested with accelerated weathering methods to assess their anticorrosive performance. The results of lab testing often do not correlate well with results from field testing, which resembles the conditions of actual use more closely. We tested the correlation of the neutral salt spray test (NSS, ISO 9227) and tests for mechanical properties and a 5-year field exposure in four different locations in the atmospheric zone, splash zone and immersion zone using 19 organic coatings for hydraulic steelwork. No correlation was found between the anticorrosive performance under outdoor exposure and the mechanical properties of a coating. The NSS test showed a positive correlation with the results in the field in 6 of 12 cases. For the fresh water testing location in Trier, the correlation proved to be very good. The biggest difference between lab and field testing was observed for zinc-primer-free coatings, which passed in the lab testing and failed in the outdoor testing. This study shows that the NSS test correlates with outdoor exposure only in some cases on a statistically significant level, but the results of NSS testing can be useful in approval testing for protective coating systems using predefined pass/fail criteria.

Improvement of RBD-FC Immunogenicity by Using Alum-Sodium Alginate Adjuvant Against SARS-COV-2.

Adjuvants use several mechanisms to boost immunogenicity and to modulate immune response. The strength of adsorption of antigen by adjuvants can be a determinant factor for significant improvement of immunopotentiation. We expressed recombinant RBD-FC in PichiaPink Strain 4 and examined the vaccination of mice by vaccine formulation with different adjuvants (sodium alginate and aluminum hydroxide, alone and together). Sodium alginate significantly increased the immunogenicity and stability of RBD-FC antigen, so RBD-FC formulated with combined alginate and alum (AlSa) and sodium alginate alone showed higher antibody titer and stability. Immunogenicity of RBD-FC:AlSa was determined by serological assays including direct enzyme-linked immunosorbent assay (ELISA) and surrogate virus neutralization test (sVNT). High levels of IgGs and neutralizing antibodies were measured in serum of mice immunized with the RBD-FC:AlSa formulation. On the other hand, cytokines IL-10 and INF-γ were severely accumulated in response to RBD-FC:AlSa, and after 10 days, their accumulation was significantly declined, whereas IL-4 showed the highest and the lowest accumulation in response to alum and alginate, respectively. Our data may suggest that combination of alum and sodium alginate has a better compatibility with RBD-FC in vaccine formulation.

Genetic and Haplotype Diversity of Clarias gariepinus (Burchell 1822) Based on Cytochrome c Oxidase I (COI) Gene

The evaluation of mitochondrial DNA and genetic analysis is helpful for economically significant species. Clarias gariepinus is a critical species in aquaculture. This study investigates the genetic diversity and population differentiation of C. gariepinus from 19 countries using 164 sequences of the mitochondrial DNA’s Cytochrome c oxidase I (COI) gene. The haplotype analysis revealed a total of 17 haplotypes, with a nucleotide diversity (π) of 0.012 and a haplotype diversity (Hd) of 0.87. The results of an AMOVA and fixation index indicated significant genetic variation and structure among the populations. Additionally, neutrality tests and mismatch distribution analysis supported the hypothesis of under-purifying selection in C. gariepinus. The findings suggested that the population did not experience expansion. In conclusion, the genetic analysis highlighted substantial variation among C. gariepinus populations from different locations, providing valuable insights for the global management of this species.

Humoral Immunity Profiling to Pandemic and Bat‐Derived Coronavirus Variants: A Geographical Comparison

AbstractDynamic pathogen exposure may impact the immunological response to SARS‐CoV‐2 (SCV2). One potential explanation for the lack of severe SCV2‐related morbidity and mortality in Southeast Asia is prior exposure to related betacoronaviruses. Recent discoveries of SCV2‐related betacoronaviruses from horseshoe bats (Rhinolophus sinicus) in Thailand, Laos, and Cambodia suggest the potential for bat‐to‐human spillover exposures in the region. In this work, serum antibodies to protein constructs from SCV2 and a representative bat coronavirus isolated in Cambodia (RshSTT182) are measured in pre‐pandemic Cambodian human sera using ELISA assays. Of 293 Cambodian samples tested (N = 131 with acute malaria, n = 162 with acute undifferentiated febrile illness), 32 (10.9%) are seropositive for SCV2 based on established Spike and receptor‐binding domain (RBD) cutoffs. Within SCV2 seropositive samples, 16 (50%) have higher antibody levels to antigens from the representative virus RshSTT182 versus SCV2 antigens; competitive binding ELISA assays demonstrate inhibition of reactivity to SCV2 Spike after pre‐incubation with RshSTT182 Spike. Surrogate virus neutralization tests demonstrate that 8/30 (26.7%) SCV2 ELISA positive pre‐pandemic Cambodian samples have neutralizing activity against SCV2, while 14/30 (46.7%) have activity against other SCV2‐related betacoronaviruses. These data suggest that exposure to related betacoronaviruses may elicit cross‐reactive immunity to SCV2 prior to the global pandemic.

Serotype-specific epidemiological patterns of inapparent versus symptomatic primary dengue virus infections: a 17-year cohort study in Nicaragua.

Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). We analysed primary DENV infections in the PDCS from 2004 to 2022 detected by inhibition ELISA (iELISA) or RT-PCR. First, we evaluated the performance of the EDIII-MMBA for serotyping with samples characterised by RT-PCR or focus reduction neutralisation test. Next, we analysed a subset of inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighbourhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analysed by serotype. Between Aug 30, 2004, and March 10, 2022, a total of 5931 DENV-naive participants were followed in the PDCS. There were 1626 primary infections (382 symptomatic, 1244 inapparent) detected by iELISA or RT-PCR over the study period. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95% CI 95·8-100) for serotyping inapparent primary DENV infections when evaluated against gold-standard serotyping methods. Of the 1244 inapparent infections, we analysed 574 (46%) using the EDIII-MMBA. We found that the majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic (pooled odds ratio compared with DENV1: 2·13, 95% CI 1·28-3·56) and severe (6·75, 2·01-22·62) primary infections, whereas DENV2 was similar to DENV1 in both analyses. Considerable within-year and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The substantial differences in infection outcomes by serotype emphasises the need for vaccines with balanced immunogenicity and efficacy across serotypes. National Institute of Allergy and Infectious Diseases (National Institutes of Health) and Bill & Melinda Gates Foundation. For the Spanish translation of the abstract see Supplementary Materials section.

Population genetic diversity of Culex tritaeniorhynchus in Jining City of Shandong Province based on the mitochondrial cytochrome C oxidase I gene

To understand the genetic basis of the adaptation of Culex tritaeniorhynchus to different environmental ecology in Jining City, Shandong Province, so as to provide insights into understanding of the population structure or isolation pattern of Cx. tritaeniorhynchus in the city. Seven sampling sites were selected from urban, suburban and rural areas of Jining City, Shandong Province from June to August 2023, and mosquitoes were collected using mosquito-trapping lamps. All collected adult mosquitoes were identified morphologically. Genomic DNA was extracted from a single female Cx. tritaeniorhynchus mosquito, and the mitochondrial cytochrome C oxidase I (COI) gene was amplified using a PCR assay, sequenced and subjected to molecular identification. The number of haplotypes, haplotype diversity (Hd), nucleotide diversity (Pi), and average number of nucleotide differences (K) of Cx. tritaeniorhynchus DNA sequences were estimated among different sampling sites using the software DnaSP 6, and a neutrality test was performed. The fixation index (FST), and gene flow (number of migrants, Nm) of Cx. tritaeniorhynchus populations were calculated using the software Arlequin 3.5.2, and subjected to analysis of molecular variance (AMOVA). In addition, a haplotype network diagrams and a phylogenetic tree of Cx. tritaeniorhynchus populations were created using the software PopART and MEGA 11, respectively. A total of 420 sequences were successfully amplified from the COI gene of Cx. tritaeniorhynchus samples collected from 7 sampling sites in Jining City, and a gene fragment sequence with a length of 603 bp was obtained, with 55 variable sites and 46 haplotypes and without insertion or deletion mutations. Of the 46 haplotypes, H01 was the dominant shared haplotype, and the haplotype frequency increased gradually from urban areas (34.00%) to rural areas (47.00%). The mean Hd, Pi and K values of Cx. tritaeniorhynchus COI genes were 0.814, 0.024 and 14.129, 0.489, 0.016 and 7.941 and 0.641, 0.016 and 10.393 in suburban, urban, and rural areas, respectively, with the highest population diversity of Cx. tritaeniorhynchus in suburban areas and the lowest in urban areas. Paired FST analysis among different types of sampling sites showed that the mean FST value was 0.029 between urban and suburban areas, indicating more frequent inter-population communication. AMOVA revealed that the percentage of intra-population variation (95.74%) was higher than that of inter-population variation (4.26%). Neutrality tests showed deviation from neutrality in Cx. tritaeniorhynchus populations collected from Nanyang Township (Tajima's D = 2.793, Fu's Fs = 6.429, both P values < 0.05). In addition, the mismatch distribution curves of Cx. tritaeniorhynchus COI gene appeared bimodal or multimodal patterns in Jining City, indicating a relatively stable overall population size. The mitochondrial COI gene may be used as a molecular marker to investigate the population genetic diversity of Cx. tritaeniorhynchus. The population genetic diversity of Cx. tritaeniorhynchus is higher in the suburban areas of Jining City than in rural and urban areas, and there are frequent genetic exchanges between Cx. tritaeniorhynchus populations from urban and suburban areas.

Development and optimization of a double antigen sandwich ELISA detecting for Senecavirus A antibodies based on VP2 protein.

Senecavirus A (SVA) is an emerging viral pathogen that threatens the global swine industry significantly. The major clinical symptoms of SVA-infected animals are vesicular lesions, but various diseases can cause the same symptoms, which makes it difficult to distinguish SVA from other vesicular diseases clinically. The absence of an effective and safe vaccine necessitates the development of a simple, specific, and sensitive serological detection method for SVA antibodies. The VP2 protein of SVA, characterized by high immunogenicity and sequence conservatism, is an essential target for serological diagnosis. In this study, a double-antigen sandwich enzyme-linked immunosorbent assay [ELISA (DAgS-ELISA)] based on VP2 protein expressed by Escherichia coli was established for SVA antibody detection. With a cut-off value of 0.237, this assay demonstrated outstanding performance, showing high sensitivity and sharp specificity, which is manifested in the absence of cross-reaction with classical swine fever virus (CSFV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV), and foot-and-mouth disease virus (FMDV) serotype A and O. Additionally, the repeatability of the method is remarkable, as shown by the coefficients variation (CV) of both the intra- and inter-assay below 10%. By detecting 166 clinical sera, it was found that the kappa value of the DAgS-ELISA was 0.78 compared with that of the virus neutralization test (VNT), indicating a high level of consistency. In general, this method has high sensitivity, sharp specificity, remarkable repeatability, sound consistency, and low cost, making it a reliable and effective tool for detecting SVA antibodies.IMPORTANCESVA has rapidly become prevalent in many countries, and its outbreaks have threatened the global swine industry significantly. The major clinical symptoms of SVA-infected animals are vesicular lesions that are similar to other vesicular diseases, making it difficult to distinguish SVA. Currently, no commercial vaccines are available for SVA; therefore, effective diagnosis of SVA infection is vital for its prevention and control. In this study, VP2 protein of SVA was expressed by E. coli, and a double-antigen sandwich enzyme-linked immunosorbent assay [ELISA (DAgS-ELISA)] for SVA antibodies detection was successfully established based on the VP2 protein. The DAgS-ELISA has a high sensitivity, sharp specificity, remarkable repeatability, sound consistency, and low cost for detecting SVA antibodies. Therefore, the DAgS-ELISA established in this study may be a reliable and effective tool for detecting SVA antibodies and may be used to strengthen the monitoring and prevention of SVA epidemic in the long run.