Overview of immunological & virological factors driving the evolution & global spread of SARS-CoV-2 variants.

The SARS-CoV-2, a highly infectious positive strand RNA virus first identified in December 2019, has produced multiple genetic variants that have rapidly and sequentially spread worldwide during the coronavirus disease 2019 (COVID-19) pandemic. Genetic changes in SARS-CoV-2 for greater infectivity, replication and transmission were selected during the early stages of the pandemic. More recently, after widespread infection and vaccination, SARS-CoV-2 variants that evade antigen-specific adaptive immunity, have begun to be selected. This article provides an overview of the molecular immunological and virological factors underlying the origin and global spread of important SARS-CoV-2 variant lineages.

Recombinant protein immunoblots for differential diagnosis of tick-borne relapsing fever and Lyme disease.

Lyme disease (LD) is caused by a group of tick-borne bacteria of the genus Borrelia termed Lyme disease Borreliae (LDB). The detection of serum antibodies to specific LDB antigens is widely used to support diagnosis of LD. Recent findings highlight a need for serological tests that can differentiate LD from tick-borne relapsing fever (TBRF) caused by a separate group of Borrelia species termed relapsing fever Borreliae. This is because LD and TBRF share some clinical symptoms and can occur in overlapping locations. The development of serological tests for TBRF is at an early stage compared with LD. This article reviews the application of line immunoblots (IBs), where recombinant proteins applied as lines on nitrocellulose membrane strips are used to detect antibodies in patient sera, for the diagnosis and differentiation of LD and TBRF.

Morphological and odorant-binding protein 1 gene intron 1 sequence variations in Anopheles stephensi from Jaffna city in northern Sri Lanka.

Three Anopheles stephensi biotypes have historically been differentiated through variations in the mode numbers of egg ridges and adult spiracular indices. Anopheles stephensi odorant-binding protein 1 gene (AsteObp1) sequences in Iran and Afghanistan have been recently interpreted to suggest that the three biotypes are sibling species. AsteObp1 intron 1 sequences, mode numbers of egg ridges and spiracular indices of An. stephensi in Jaffna city in Sri Lanka were therefore investigated in field-collected mosquitoes and short-term laboratory colonies established from them. AsteObp1 intron 1 sequences revealed the region to be polymorphic with four unique sequences, ASJF1-4, present in both short-term laboratory colonies and field-collected An. stephensi. The spiracular index did not relate to the mode number of egg ridges in Jaffna An. stephensi. The results suggested that numbers of egg ridges, spiracular indices and AsteObp1 intron 1 sequences were not useful for differentiating An. stephensi biotypes in Jaffna. It is proposed that the observed differences between An. stephensi mosquitoes in Jaffna now result from normal population variance in the context of rapidly changing bionomics in India and northern Sri Lanka.

Transcriptomic, proteomic and ultrastructural studies on salinity-tolerant Aedes aegypti in the context of rising sea levels and arboviral disease epidemiology.

BACKGROUND: Aedes aegypti mosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance in Ae. aegypti has not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity. RESULTS: BW- and FW-Ae. aegypti were compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerant Ae. aegypti were also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults. CONCLUSIONS: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerant Ae. aegypti are expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerant Ae. aegypti and genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerant Ae. aegypti and requires appropriate mitigating measures. The findings in Ae. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.

IgG and IgM antibody formation to spike and nucleocapsid proteins in COVID-19 characterized by multiplex immunoblot assays.

BACKGROUND: Rapid and simple serological assays for characterizing antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiplex immunoblot (IB) assays termed COVID-19 IB assays were developed for detecting IgG and IgM antibodies to SARS-CoV-2 virus proteins in COVID-19 patients. METHODS: Recombinant nucleocapsid protein and the S1, S2 and receptor binding domain (RBD) of the spike protein of SARS-CoV-2 were used as target antigens in the COVID-19 IBs. Specificity of the IB assay was established with 231 sera from persons with allergy, unrelated viral infections, autoimmune conditions and suspected tick-borne diseases, and 32 goat antisera to human influenza proteins. IgG and IgM COVID-19 IBs assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms. RESULTS: Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of the COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for either IgG or IgM antibodies meet the US recommendations for laboratory serological diagnostic tests. The proportion of IgM-positive sera from the COVID-19 patients following an RT-qPCR positive test was maximal at 83% before 10 days and decreased to 0% after 100 days, while the proportions of IgG-positive sera tended to plateau between days 11 and 65 at 78-100% and fall to 44% after 100 days. Detection of either IgG or IgM antibodies was better than IgG or IgM alone for assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than S1, S2 and N proteins. CONCLUSIONS: The multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.

Perspective of the Relationship between the Susceptibility to Initial SARS-CoV-2 Infectivity and Optimal Nasal Conditioning of Inhaled Air.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as with the influenza virus, has been shown to spread more rapidly during winter. Severe coronavirus disease 2019 (COVID-19), which can follow SARS-CoV-2 infection, disproportionately affects older persons and males as well as people living in temperate zone countries with a tropical ancestry. Recent evidence on the importance of adequately warming and humidifying (conditioning) inhaled air in the nasal cavity for reducing SARS-CoV-2 infectivity in the upper respiratory tract (URT) is discussed, with particular reference to: (i) the relevance of air-borne SARS-CoV-2 transmission, (ii) the nasal epithelium as the initial site of SARS-CoV-2 infection, (iii) the roles of type 1 and 3 interferons for preventing viral infection of URT epithelial cells, (iv) weaker innate immune responses to respiratory viral infections in URT epithelial cells at suboptimal temperature and humidity, and (v) early innate immune responses in the URT for limiting and eliminating SARS-CoV-2 infections. The available data are consistent with optimal nasal air conditioning reducing SARS-CoV-2 infectivity of the URT and, as a consequence, severe COVID-19. Further studies on SARS-CoV-2 infection rates and viral loads in the nasal cavity and nasopharynx in relation to inhaled air temperature, humidity, age, gender, and genetic background are needed in this context. Face masks used for reducing air-borne virus transmission can also promote better nasal air conditioning in cold weather. Masks can, thereby, minimise SARS-CoV-2 infectivity and are particularly relevant for protecting more vulnerable persons from severe COVID-19.

Population genomics reveals that an anthropophilic population of Aedes aegypti mosquitoes in West Africa recently gave rise to American and Asian populations of this major disease vector.

BACKGROUND: The mosquito Aedes aegypti is the main vector of dengue, Zika, chikungunya and yellow fever viruses. This major disease vector is thought to have arisen when the African subspecies Ae. aegypti formosus evolved from being zoophilic and living in forest habitats into a form that specialises on humans and resides near human population centres. The resulting domestic subspecies, Ae. aegypti aegypti, is found throughout the tropics and largely blood-feeds on humans. RESULTS: To understand this transition, we have sequenced the exomes of mosquitoes collected from five populations from around the world. We found that Ae. aegypti specimens from an urban population in Senegal in West Africa were more closely related to populations in Mexico and Sri Lanka than they were to a nearby forest population. We estimate that the populations in Senegal and Mexico split just a few hundred years ago, and we found no evidence of Ae. aegypti aegypti mosquitoes migrating back to Africa from elsewhere in the tropics. The out-of-Africa migration was accompanied by a dramatic reduction in effective population size, resulting in a loss of genetic diversity and rare genetic variants. CONCLUSIONS: We conclude that a domestic population of Ae. aegypti in Senegal and domestic populations on other continents are more closely related to each other than to other African populations. This suggests that an ancestral population of Ae. aegypti evolved to become a human specialist in Africa, giving rise to the subspecies Ae. aegypti aegypti. The descendants of this population are still found in West Africa today, and the rest of the world was colonised when mosquitoes from this population migrated out of Africa. This is the first report of an African population of Ae. aegypti aegypti mosquitoes that is closely related to Asian and American populations. As the two subspecies differ in their ability to vector disease, their existence side by side in West Africa may have important implications for disease transmission.

Mosquito vectors developing in atypical anthropogenic habitats: Global overview of recent observations, mechanisms and impact on disease transmission.

The major mosquito vectors of human diseases have co-evolved with humans over a long period of time. However, the rapid growth in human population and the associated expansion in agricultural activity and greater urbanisation have created ecological changes that have had a marked impact on biology of mosquito vectors. Adaptation of the vectors of malaria and important arbovial diseases over a much shorter time scale to the new types of preimaginal habitats recently created by human population growth and activity is highlighted here in the context of its potential for increasing disease transmission rates. Possible measures that can reduce the effects on the transmission of mosquito-borne diseases are also outlined.

Bionomic aspects of the Anopheles subpictus species complex in Sri Lanka.

Anopheles subpictus Grassi s.l. (Diptera: Culicidae) functions as a secondary malaria vector to Anopheles culicifacies Giles s.l. (Diptera: Culicidae) in Sri Lanka. The taxon A. subpictus is reported to exist as a species complex comprising four sibling species (A-D) that can be differentiated through polytene chromosome banding patterns and stage-specific morphometric traits in India. Based on the morphological characteristics described for the Indian Subpictus Complex, the presence of all four sibling species has been described in Sri Lanka. As sibling species show distinct bio-ecological characteristics that are important for devising appropriate vector control measures, a study was carried out in six districts in the dry zone of Sri Lanka. The results confirm the presence of all four sibling species, with species C predominating in inland areas and species B in coastal areas. Species C and D were indoor-resting and indoor-feeding, while species B was outdoor-resting with no significant preference for indoor- or outdoor-resting. Species B showed distinct morphological variation in the ornamentation of wings and palpi. Blood meal analysis revealed that species B, C, and D can feed on humans as well as cattle. The differential bio-ecological traits shown by the members of the Subpictus Complex are important for developing appropriate vector control measures in Sri Lanka.

Target Antigens in Western and Line Immunoblots for Supporting the Diagnosis of Lyme Disease. Comment on Porwancher et al. Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches. Pathogens 2023, 12, 1282.

An article was recently published in Pathogens on using different target antigens from Borrelia species that cause Lyme disease for detecting serum antibodies to support a clinical diagnosis of Lyme disease (LD) [...].

Robustness in population-structure and demographic-inference results derived from the Aedes aegypti genotyping chip and whole-genome sequencing data.

The mosquito Aedes aegypti is the primary vector of many human arboviruses such as dengue, yellow fever, chikungunya, and Zika, which affect millions of people worldwide. Population genetic studies on this mosquito have been important in understanding its invasion pathways and success as a vector of human disease. The Axiom aegypti1 SNP chip was developed from a sample of geographically diverse A. aegypti populations to facilitate genomic studies on this species. We evaluate the utility of the Axiom aegypti1 SNP chip for population genetics and compare it with a low-depth shotgun sequencing approach using mosquitoes from the native (Africa) and invasive ranges (outside Africa). These analyses indicate that results from the SNP chip are highly reproducible and have a higher sensitivity to capture alternative alleles than a low-coverage whole-genome sequencing approach. Although the SNP chip suffers from ascertainment bias, results from population structure, ancestry, demographic, and phylogenetic analyses using the SNP chip were congruent with those derived from low-coverage whole-genome sequencing, and consistent with previous reports on Africa and outside Africa populations using microsatellites. More importantly, we identified a subset of SNPs that can be reliably used to generate merged databases, opening the door to combined analyses. We conclude that the Axiom aegypti1 SNP chip is a convenient, more accurate, low-cost alternative to low-depth whole-genome sequencing for population genetic studies of A. aegypti that do not rely on full allelic frequency spectra. Whole-genome sequencing and SNP chip data can be easily merged, extending the usefulness of both approaches.

Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka.

BACKGROUND: Anopheles subpictus sensu lato is a major malaria vector in South and Southeast Asia. Based initially on polytene chromosome inversion polymorphism, and subsequently on morphological characterization, four sibling species A-D were reported from India. The present study uses molecular methods to further characterize and identify sibling species in Sri Lanka. METHODS: Mosquitoes from Sri Lanka were morphologically identified to species and sequenced for the ribosomal internal transcribed spacer-2 (ITS2) and the mitochondrial cytochrome c oxidase subunit-I (COI) genes. These sequences, together with others from GenBank, were used to construct phylogenetic trees and parsimony haplotype networks and to test for genetic population structure. RESULTS: Both ITS2 and COI sequences revealed two divergent clades indicating that the Subpictus complex in Sri Lanka is composed of two genetically distinct species that correspond to species A and species B from India. Phylogenetic analysis showed that species A and species B do not form a monophyletic clade but instead share genetic similarity with Anopheles vagus and Anopheles sundaicus s.l., respectively. An allele specific identification method based on ITS2 variation was developed for the reliable identification of species A and B in Sri Lanka. CONCLUSION: Further multidisciplinary studies are needed to establish the species status of all chromosomal forms in the Subpictus complex. This study emphasizes the difficulties in using morphological characters for species identification in An. subpictus s.l. in Sri Lanka and demonstrates the utility of an allele specific identification method that can be used to characterize the differential bio-ecological traits of species A and B in Sri Lanka.

Predatory efficacy of Culex (Lutzia) fuscanus on mosquito vectors of human diseases in Sri Lanka.

Larvae of Culex (Lutzia) Fuscanus were collected from ovitraps in a natural breeding site. Collected larvae were used to establish a self-mating colony, and larval progeny were then used to determine their predatory efficacy on larvae of 3 vector mosquito species, Aedes aegypti, Anopheles subpictus, and Cx. tritaeniorhynchus. Statistical analysis revealed that Cx. fuscanus showed greater feeding efficacy for Ae. aegypti than for Cx. tritaeniorhynchus and An. subpictus. The natural predatory role of this species can potentially be exploited for biological control of mosquito vectors in Sri Lanka.

COVID-19 Vaccines for Optimizing Immunity in the Upper Respiratory Tract.

Rapid development and deployment of vaccines greatly reduced mortality and morbidity during the COVID-19 pandemic. The most widely used COVID-19 vaccines approved by national regulatory authorities require intramuscular administration. SARS-CoV-2 initially infects the upper respiratory tract, where the infection can be eliminated with little or no symptoms by an effective immune response. Failure to eliminate SARS-CoV-2 in the upper respiratory tract results in lower respiratory tract infections that can lead to severe disease and death. Presently used intramuscularly administered COVID-19 vaccines are effective in reducing severe disease and mortality, but are not entirely able to prevent asymptomatic and mild infections as well as person-to-person transmission of the virus. Individual and population differences also influence susceptibility to infection and the propensity to develop severe disease. This article provides a perspective on the nature and the mode of delivery of COVID-19 vaccines that can optimize protective immunity in the upper respiratory tract to reduce infections and virus transmission as well as severe disease.

Resistance to the larvicide temephos and altered egg and larval surfaces characterize salinity-tolerant Aedes aegypti.

Aedes aegypti, the principal global vector of arboviral diseases and previously considered to oviposit and undergo preimaginal development only in fresh water, has recently been shown to be capable of developing in coastal brackish water containing up to 15 g/L salt. We investigated surface changes in eggs and larval cuticles by atomic force and scanning electron microscopy, and larval susceptibility to two widely-used larvicides, temephos and Bacillus thuringiensis, in brackish water-adapted Ae. aegypti. Compared to freshwater forms, salinity-tolerant Ae. aegypti had rougher and less elastic egg surfaces, eggs that hatched better in brackish water, rougher larval cuticle surfaces, and larvae more resistant to the organophosphate insecticide temephos. Larval cuticle and egg surface changes in salinity-tolerant Ae. aegypti are proposed to respectively contribute to the increased temephos resistance and egg hatchability in brackish water. The findings highlight the importance of extending Aedes vector larval source reduction efforts to brackish water habitats and monitoring the efficacy of larvicides in coastal areas worldwide.

Terminal galactosylation of glycoconjugates in Plasmodium falciparum asexual blood stages and Trypanosoma brucei bloodstream trypomastigotes.

There is definitive biochemical evidence for the presence of terminal α-galactosyl residues (α-gal) in the N-linked oligosaccharides and glycophosphatidylinositol anchors (GPI anchors) of the variant surface glycoprotein of Trypanosoma brucei bloodstream trypomastigotes. Indirect evidence also exists for α-gal in Plasmodium falciparum asexual blood stage glycoproteins and glycolipids. The occurrence of α-gal in glycoproteins and glycolipids of T. brucei bloodstream trypomastigotes and P. falciparum late asexual blood stages was investigated by the binding of α-gal-specific Bandeirea simplicifolia B4 lectin 1 (BSB4), incorporation of [(3)H]galactose from UDP-[(3)H]galactose into glycoproteins and glycolipids in microsomes in vitro, and bioinformatic searches for galactosyl-transferase coding sequences. The findings confirm the presence of α-gal in a spectrum of T. brucei bloodstream trypomastigote glycoproteins and glycolipids and indicate its relative absence from P. falciparum asexual blood stage glycoconjugates.

Innate and Adaptive Immune Responses in the Upper Respiratory Tract and the Infectivity of SARS-CoV-2.

Increasing evidence shows the nasal epithelium to be the initial site of SARS-CoV-2 infection, and that early and effective immune responses in the upper respiratory tract (URT) limit and eliminate the infection in the URT, thereby preventing infection of the lower respiratory tract and the development of severe COVID-19. SARS-CoV-2 interferes with innate immunity signaling and evolves mutants that can reduce antibody-mediated immunity in the URT. Recent genetic and immunological advances in understanding innate immunity to SARS-CoV-2 in the URT, and the ability of prior infections as well as currently available injectable and potential intranasal COVID-19 vaccines to generate anamnestic adaptive immunity in the URT, are reviewed. It is suggested that the more detailed investigation of URT immune responses to all types of COVID-19 vaccines, and the development of safe and effective COVID-19 vaccines for intranasal administration, are important needs.

Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases.

Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.

Dengue Incidence and Aedes Vector Collections in Relation to COVID-19 Population Mobility Restrictions.

Contrary to expectation, dengue incidence decreased in many countries during the period when stringent population movement restrictions were imposed to combat COVID-19. Using a seasonal autoregressive integrated moving average model, we previously reported a 74% reduction in the predicted number of dengue cases from March 2020 to April 2021 in the whole of Sri Lanka, with reductions occurring in all 25 districts in the country. The reduction in dengue incidence was accompanied by an 87% reduction in larval collections of Aedes vectors in the northern city of Jaffna. It was proposed that movement restrictions led to reduced human contact and blood feeding by Aedes vectors, accompanied by decreased oviposition and vector densities, which were responsible for diminished dengue transmission. These findings are extended in the present study by investigating the relationship between dengue incidence, population movement restrictions, and vector larval collections between May 2021 and July 2022, when movement restrictions began to be lifted, with their complete removal in November 2021. The new findings further support our previous proposal that population movement restrictions imposed during the COVID-19 pandemic reduced dengue transmission primarily by influencing human-Aedes vector interaction dynamics.

Possible impact of rising sea levels on vector-borne infectious diseases.

BACKGROUND: Vector-borne infectious diseases are a significant cause of human and animal mortality and morbidity. Modeling studies predict that changes in climate that accompany global warming will alter the transmission risk of many vector-borne infectious diseases in different parts of the world. Global warming will also raise sea levels, which will lead to an increase in saline and brackish water bodies in coastal areas. The potential impact of rising sea levels, as opposed to climate change, on the prevalence of vector-borne infectious diseases has hitherto been unrecognised. PRESENTATION OF THE HYPOTHESIS: Mosquito species possessing salinity-tolerant larvae and pupae, and capable of transmitting arboviruses and parasites are found in many parts of the world. An expansion of brackish and saline water bodies in coastal areas, associated with rising sea levels, can increase densities of salinity-tolerant vector mosquitoes and lead to the adaptation of freshwater vectors to breed in brackish and saline waters. The breeding of non-mosquito vectors may also be influenced by salinity changes in coastal habitats. Higher vector densities can increase transmission of vector-borne infectious diseases in coastal localities, which can then spread to other areas. TESTING THE HYPOTHESIS: The demonstration of increases in vector populations and disease prevalence that is related to an expansion of brackish/saline water bodies in coastal areas will provide the necessary supportive evidence. However the implementation of specific vector and disease control measures to counter the threat will confound the expected findings. IMPLICATIONS OF THE HYPOTHESIS: Rising sea levels can act synergistically with climate change and then interact in a complex manner with other environmental and socio-economic factors to generate a greater potential for the transmission of vector-borne infectious diseases. The resulting health impacts are likely to be particularly significant in resource-poor countries in the tropics and semi-tropics. Some measures to meet this threat are outlined.