Antigenic determinants of HAstV-VA1 neutralization and their relevance in the human immune response.

Astroviruses are highly divergent and infect a wide variety of animal hosts. In 2009, a genetically divergent human astrovirus (HAstV) strain VA1 was first identified in an outbreak of acute gastroenteritis. This strain has also been associated with fatal central nervous system disease. In this work, we report the isolation of three high-affinity neutralizing monoclonal antibodies (Nt-MAbs) targeting the capsid spike domain of HAstV-VA1. These antibodies (7C8, 2A2, 3D8) were used to select individual HAstV-VA1 mutants resistant to their neutralizing activity and also select a HAstV-VA1 triple mutant that escapes neutralization from all three Nt-MAbs. Sequencing of the virus genome capsid region revealed escape mutations that map to the surface of the capsid spike domain, define three potentially independent neutralization epitopes, and help delineate four antigenic sites in rotaviruses. Notably, two of the escape mutations were found to be present in the spike sequence of the HAstV-VA1-PS strain isolated from an immunodeficient patient with encephalitis, suggesting that those mutations arose as a result of the immune pressure generated by the patient's immunotherapy. In accordance with this observation, human serum samples exhibiting strong neutralization activity against wild-type HAstV-VA1 had a 2.6-fold reduction in neutralization titer when evaluated against the triple-escape HAstV-VA1 mutant, indicating shared neutralization epitopes between the mouse and human antibody response. The isolated Nt-MAbs reported in this work will help characterize the functional sites of the virus during cell entry and have the potential for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1. Importance: Human astroviruses (HAstVs) have been historically associated with acute gastroenteritis. However, the genetically divergent HAstV-VA1 strain has been associated with central nervous system disease. This work isolated high-affinity neutralizing monoclonal antibodies directed to HAstV-VA1. The proposed binding sites for these antibodies, together with previously reported sites for neutralizing antibodies against classical HAstVs, suggest the existence of at least four neutralization sites on the capsid spike of astroviruses. Our data show that natural infection with human astrovirus VA1 elicits a robust humoral immune response that targets the same antigenic sites recognized by the mouse monoclonal antibodies and strongly suggests the emergence of a variant HAstV-VA1 virus in an immunodeficient patient with prolonged astrovirus infection. The isolated Nt-MAb reported in this work will be helpful in defining the functional sites of the virus involved in cell entry and hold promise for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1.

Structure and antigenicity of the divergent human astrovirus VA1 capsid spike.

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

Cross-reactivity of SARS-CoV-2 with other pathogens, especially dengue virus: A historical perspective.

Dengue is a vector-borne viral disease caused by a Flavivirus whereas the COVID-19 pandemic was caused by a highly contagious virus, SARS-CoV-2 belonging to the family Coronaviridae. However, COVID-19 severity was observably less in dengue-endemic countries and vice versa especially during the active years of the pandemic (2019-2021). We observed that dengue virus (DENV) antibodies (Abs) could cross-react with SARS-CoV-2 spike antigen. This resulted in SARS-CoV-2 false positivity by rapid Ab test kits. DENV Abs binding to SARS-CoV-2 receptor-binding domain (and the reverse scenario), as revealed by docking studies further validated DENV and SARS-CoV-2 cross-reactivity. Finally, SARS-CoV-2 Abs were found to cross-neutralize DENV1 and DENV2 in virus neutralization test (VNT). Abs to other pathogens like Plasmodium were also cross-reactive but non-neutralizing for SARS-CoV-2. Here, we analyze the existing data on SARS-CoV-2 cross-reactivity with other pathogens, especially dengue to assess its impact on health (cross-protection?) and differential sero-diagnosis/surveillance.

Erratum: Author affiliation addition: "Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?"

[This corrects the article on p. 775 in vol. 12, PMID: 33200016.].

Non-structural protein 1 (NS1) variants from dengue virus clinical samples revealed mutations that influence NS1 production and secretion.

Dengue diagnosis primarily relies on NS1 ELISA and serological (IgG/IgM) tests. There are reports of low and variable sensitivity of the widely used NS1 ELISA tests. Poor sensitivity has been attributed to patient's infection status, prevalent serotypes, and the geographical origin of the samples. We investigated whether NS1 mutations directly have any impact on NS1 ELISA-based dengue virus (DENV) detection in clinical samples. Fifty-eight serum samples were collected from dengue-endemic area during 2015-2017 and tested with three commonly used NS1 ELISA kits. The samples were subjected to diagnostic RT-PCR and sequencing of structural gene(s). Sequencing of NS1 gene revealed amino acid changes which were transferred to respective wild type NS1 backbone to determine their effects on NS1 production and secretion in Huh-7, Vero, and A549 cells. Eighty-seven percent samples were virus RNA-positive but 65% of these were NS1 ELISA-positive. NS1-gene mutations like Val236➔Ala (DENV2) or Trp68➔stop codon in DENV3 were associated with decreased NS1 production and secretion. These mutations were originally identified in NS1 ELISA-negative clinical isolates. All DENV1 and > 80% DENV2 were NS1 ELISA-positive. The three NS1 ELISA could not detect recently circulating DENV3 single infections despite being RNA-positive. Among serotypes 1-3, wild-type NS1 production was highest for DENV1 and lowest for DENV3 in all cell lines tested. Mutations in circulating DENV directly correlated with NS1 production and secretion and, hence, ELISA-based NS1 detection. Further studies to define more NS1 mutations in clinical samples are needed to optimize ELISA kits for more sensitive dengue diagnosis.

Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?

BACKGROUND: The recent rise in the incidence of hepatitis B virus (HBV) infections in a densely populated city of eastern India ("mixing vessel" of people of varied socio-economic and immune status) prompted this study. Applying saliva on fingers for enumerating bank notes is a common practice in the Indian subcontinent. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin. AIM: To investigate whether paper currencies could be a plausible mode of horizontal transmission of HBV infection. METHODS: Polymerase chain reactions (PCR) followed by nucleotide sequencing was done for the detection of HBV. Hepatitis B virus surface antigen enzyme-linked immunosorbent assay(HBsAg ELISA) was performed on all HBV deoxyribonucleic acid-positive samples to check the detectability of the virus. Atomic force microscopy (AFM) was carried out for visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. RESULTS: HBV-specific PCRs on pellets obtained after ultracentrifugation/ immunoprecipitation of the currency paper washings detected potentially intact/viable HBV (genotype D2) in 7.14% of samples (n = 70). AFM gave the visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. However, HBV isolates from the currency notes could not be detected by HBsAg ELISA. CONCLUSION: It is a common practice in the Indian subcontinent to count paper currencies by applying saliva on fingertips. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin, but it was practically not possible to demonstrate experimentally such transmission. Detection of potentially intact/viable and "occult" HBV from currency poses potential risk of silent transmission of this virus among the general population.

In-depth molecular analysis of a small cohort of human and Aedes mosquito (adults and larvae) samples from Kolkata revealed absence of Zika but high prevalence of dengue virus.

PURPOSE: Zika virus infections have recently been reported in many dengue-endemic areas globally. Both dengue (DENV) and Zika (ZIKV) virus are transmitted by Aedes mosquitoes, raising the possibility of mixed infections in both vector and host. We evaluated DENV and ZIKV prevalence in human and vector samples in Kolkata, a DENV-endemic city. METHODOLOGY: Blood samples were collected from 70 patients presenting dengue-like fever symptoms at a hospital in Kolkata during 2015-16. Serum was obtained and tested for DENV infection by DENV NS1-based ELISA. Adult (n=8) and larval stages (n=12) of Aedes were also collected. A RT-PCR-based screening of both viruses supplemented by amplicon sequencing was performed. RESULTS: Of the 70 samples, 20 DENV NS1-positive serum samples were used for detailed molecular study for DENV infection. Eighteen of these (90 %) were positive by hemi-nested serotype-specific RT-PCR for DENV1/2/3, with four samples showing evidence of DENV2-3 or DENV1-3 mixed infection. None were ZIKV-positive using NS5 or ENV-based PCR, though weak amplification of a DENV1 NS5 sequence was detected in three serum samples indicating cross-reactivity of the primers. All mosquito samples were ZIKV-negative, whereas 5/8 (63 %) of adult mosquitoes and 11/12 (92 %) of larvae were DENV3-positive. CONCLUSION: Both host and vector samples showed absence of ZIKV but high prevalence of DENV. The high rate of infection of larvae with DENV is suggestive of trans-ovarial transmission that could contribute to the surge of human infections during each post-monsoon season. It would be important to guard against false positives using the available Zika-reporting primer sets.

The Non-native Helical Intermediate State May Accumulate at Low pH in the Folding and Aggregation Landscape of the Intestinal Fatty Acid Binding Protein.

There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly ß-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely ß-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein, while an identical change, distant from the hydrophobic core, does not show any effect. This study re-emphasizes an overlap between the folding and aggregation landscape of a protein, where the fine-tuning between the local and global effects may be important for the protein to fold efficiently or to aggregate.