Assessment of the broad-spectrum host targeting antiviral efficacy of halofuginone hydrobromide in human airway, intestinal and brain organotypic models.

Halofuginone hydrobromide has shown potent antiviral efficacy against a variety of viruses such as SARS-CoV-2, dengue, or chikungunya virus, and has, therefore, been hypothesized to have broad-spectrum antiviral activity. In this paper, we tested this broad-spectrum antiviral activity of Halofuginone hydrobomide against viruses from different families (Picornaviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, and Flaviviridae). To this end, we used relevant human models of the airway and intestinal epithelium and regionalized neural organoids. Halofuginone hydrobomide showed antiviral activity against SARS-CoV-2 in the airway epithelium with no toxicity at equivalent concentrations used in human clinical trials but not against any of the other tested viruses.

Non-Polio Enterovirus C Replicate in Both Airway and Intestine Organotypic Cultures.

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection. Importance: With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

Differences in systemic and mucosal SARS-CoV-2 antibody prevalence in a prospective cohort of Dutch children.

Background: As SARS-CoV-2 will likely continue to circulate, low-impact methods become more relevant to monitor antibody-mediated immunity. Saliva sampling could provide a non-invasive method with reduced impact on children. Studies reporting on the differences between systemic and mucosal humoral immunity to SARS-CoV-2 are inconsistent in adults and scarce in children. These differences may be further unraveled by exploring associations to demographic and clinical variables. Methods: To evaluate the use of saliva antibody assays, we performed a cross-sectional cohort study by collecting serum and saliva of 223 children attending medical services in the Netherlands (irrespective of SARS-CoV-2 exposure, symptoms or vaccination) from May to October 2021. With a Luminex and a Wantai assay, we measured prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD) and nucleocapsid-specific IgG and IgA in serum and saliva and explored associations with demographic variables. Findings: The S-specific IgG prevalence was higher in serum 39% (95% CI 32 - 45%) than in saliva 30% (95% CI 24 - 36%) (P ≤ 0.003). Twenty-seven percent (55/205) of children were S-specific IgG positive in serum and saliva, 12% (25/205) were only positive in serum and 3% (6/205) only in saliva. Vaccinated children showed a higher concordance between serum and saliva than infected children. Odds for saliva S-specific IgG positivity were higher in girls compared to boys (aOR 2.63, P = 0.012). Moreover, immunocompromised children showed lower odds for S- and RBD-specific IgG in both serum and saliva compared to healthy children (aOR 0.23 - 0.25, P ≤ 0.050). Conclusions: We showed that saliva-based antibody assays can be useful for identifying SARS-CoV-2 humoral immunity in a non-invasive manner, and that IgG prevalence may be affected by sex and immunocompromisation. Differences between infection and vaccination, between sexes and between immunocompromised and healthy children should be further investigated and considered when choosing systemic or mucosal antibody measurement.

Parechovirus A Infection of the Intestinal Epithelium: Differences Between Genotypes A1 and A3.

Human parechovirus (PeV-A), one of the species within the Picornaviridae family, is known to cause disease in humans. The most commonly detected genotypes are PeV-A1, associated with mild gastrointestinal disease in young children, and PeV-A3, linked to severe disease with neurological symptoms in neonates. As PeV-A are detectable in stool and nasopharyngeal samples, entry is speculated to occur via the respiratory and gastro-intestinal routes. In this study, we characterized PeV-A1 and PeV-A3 replication and tropism in the intestinal epithelium using a primary 2D model based on human fetal enteroids. This model was permissive to infection with lab-adapted strains and clinical isolates of PeV-A1, but for PeV-A3, infection could only be established with clinical isolates. Replication was highest with infection established from the basolateral side with apical shedding for both genotypes. Compared to PeV-A1, replication kinetics of PeV-A3 were slower. Interestingly, there was a difference in cell tropism with PeV-A1 infecting both Paneth cells and enterocytes, while PeV-A3 infected mainly goblet cells. This difference in cell tropism may explain the difference in replication kinetics and associated disease in humans.

Saliva SARS-CoV-2 Antibody Prevalence in Children.

COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Neutralising Antibodies against Enterovirus and Parechovirus in IVIG Reflect General Circulation: A Tool for Sero-Surveillance.

Non-polio enteroviruses (NPEV) and parechoviruses (PeV) are widespread pathogens that cause significant morbidity. Surveillance is based on culturing or genotyping of virus strains found in clinical samples. Sero-surveillance, by measuring neutralising antibodies (nAb) through virus neutralisation assays (VNA), could provide additional information as it offers a more comprehensive overview of exposure to circulating types in the general population. In our study we evaluated Intravenous immunoglobulins (IVIG) to generate sero-surveillance data. We performed VNA of nineteen NPEV and PeV with Dutch IVIG batches from two different time points (2010 and 2017) and an IVIG batch from Vietnam (2011). We compared our findings with geno- and sero-surveillance data and evaluated changes over time and between the two countries. Our findings show a good correlation with what is known from geno-surveillance data. The highest nAb titres were found against strains from Enterovirus B, while we did not observe nAb titres against strains belonging to Enterovirus C. In conclusion, we demonstrated that sero-surveillance by means of IVIG can be used to obtain insight into circulation of EV and PeV genotypes. This is of particular interest for public health, to evaluate changes over time and population susceptibility to emerging genotypes.

Seroepidemiology of Parechovirus A3 Neutralizing Antibodies, Australia, the Netherlands, and United States.

Recent parechovirus A3 (PeV-A3) outbreaks in Australia suggest lower population immunity compared with regions that have endemic PeV-A3 circulation. A serosurvey among populations in the Netherlands, the United States, and Australia before and after the 2013 Australia outbreak showed high PeV-A3 neutralizing antibody prevalence across all regions and time periods, indicating widespread circulation.

Polarized Entry of Human Parechoviruses in the Airway Epithelium.

Human parechoviruses (HPeVs), a poorly studied genus within the Picornaviridae family, are classified into 19 genotypes of which HPeV1 and HPeV3 are the most often detected. HPeV1 VP1 C terminus contains an arginine-glycine-aspartic acid (RGD) motif and has been shown to depend on the host cell surface αV integrins (αV ITGs) and heparan sulfate (HS) for entry. HPeV3 lacks this motif and the receptors remain unknown. HPeVs can be detected in patient nasopharyngeal and stool samples, and infection is presumed to occur after respiratory or gastro-intestinal transmission. HPeV pathogenesis is poorly understood as there are no animal models and previous studies have been conducted in immortalized monolayer cell cultures which do not adequately represent the characteristics of human tissues. To bridge this gap, we determined the polarity of infection, replication kinetics, and cell tropism of HPeV1 and HPeV3 in the well-differentiated human airway epithelial (HAE) model. We found the HAE cultures to be permissive for HPeVs. Both HPeV genotypes infected the HAE preferentially from the basolateral surface while the progeny virus was shed toward the apical side. Confocal microscopy revealed the target cell type to be the p63+ basal cells for both viruses, αV ITG and HS blocking had no effect on the replication of either virus, and transcriptional profiling suggested that HPeV3 infection induced stronger immune activation than HPeV1. Genotype-specific host responses may contribute to the differences in pathogenesis and clinical outcomes associated with HPeV1 and HPeV3.

Strain-dependent neutralization reveals antigenic variation of human parechovirus 3.

Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.

Prediction of Protection against Asian Enterovirus 71 Outbreak Strains by Cross-neutralizing Capacity of Serum from Dutch Donors, The Netherlands.

Outbreaks of human enterovirus 71 (EV-71) in Asia are related to high illness and death rates among children. To gain insight into the potential threat for the population of Europe, we determined the neutralizing activity in intravenous immunoglobulin (IVIg) batches and individual serum samples from donors in the Netherlands against EV-71 strains isolated in Europe and in Asia. All IVIg batches and 41%, 79%, and 65% of serum samples from children ≤5 years of age, women of childbearing age, and HIV-positive men, respectively, showed high neutralizing activity against a Dutch C1 strain, confirming widespread circulation of EV-71 in the Netherlands. Asian B3-4 and C4 strains were efficiently cross-neutralized, predicting possible protection against extensive circulation and associated outbreaks of those types in Europe. However, C2 and C5 strains that had few mutations in the capsid region consistently escaped neutralization, emphasizing the importance of monitoring antigenic diversity among circulating EV-71 strains.

Evaluation of a rapid antigen detection point-of-care test for respiratory syncytial virus and influenza in a pediatric hospitalized population in the Netherlands.

This pilot study evaluates the diagnostic performance of Sofia RSV Fluorescent Immunoassay Analyzer (FIA) and Sofia Influenza A + B FIA for rapid detection of respiratory syncytial virus and influenza A and B. Sofia had a lower-than-expected sensitivity for all viruses and a high rate of false-positive results for influenza B virus.

Pleurodynia caused by an echovirus 1 brought back from the tropics.

A 31 year-old woman presented with acute pain on the left side of the thorax and abdomen, radiating to the back together with fever, after she had returned from traveling in Southeast Asia. Except for pleural friction rub auscultated on the left hemithorax, no physical abnormalities were detected. We diagnosed a classical course of Bornholm disease, caused by an echovirus type 1. While described as a classical pathogen causing Bornholm disease, this genotype has not been reported frequently in Surveillance data in the Western World.

Systemic tryptophan and kynurenine catabolite levels relate to severity of rhinovirus-induced asthma exacerbation: a prospective study with a parallel-group design.

BACKGROUND: Patients with allergic asthma have exacerbations which are frequently caused by rhinovirus infection. The antiviral tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO) is induced by interferon-γ and suppressed by Th2 mediators interleukin (IL)-4 and IL-13. We hypothesised that local IDO activity after viral airway infection is lower in patients with allergic asthma than in healthy controls. OBJECTIVE: To determine whether IDO activity differs between patients with allergic asthma and healthy individuals before and after rhinovirus infection. METHODS: Healthy individuals and patients with allergic asthma were experimentally infected with low-dose (10 TCID50) rhinovirus 16. Blood, bronchoalveolar lavage fluid and exhaled breath condensate (for mass spectrometry by UPLC-MS/MS) were obtained before and after rhinovirus challenge. RESULTS: IDO activity was not induced by rhinovirus infection in either group, despite increases in cold scores. However, baseline pulmonary IDO activity was lower in patients with allergic asthma than in healthy individuals. In contrast, systemic tryptophan and its catabolites were markedly higher in patients with allergic asthma. Moreover, systemic quinolinic acid and tryptophan were associated with eosinophil cationic protein (r=0.43 and r=0.78, respectively) and eosinophils (r=0.38 and r=0.58, respectively) in bronchoalveolar lavage fluid and peak asthma symptom scores after rhinovirus challenge (r=0.53 and r=0.64, respectively). CONCLUSIONS: Rhinovirus infection by itself induces no IDO activity, but the reduced pulmonary IDO activity in patients with allergic asthma at baseline may underlie a reduced control of viral infections. Notably, the enhanced systemic catabolism of tryptophan in patients with allergic asthma was strongly related to the outcome of rhinovirus challenge in asthma and may serve as a prognostic factor.

Detection of human enterovirus and human parechovirus (HPeV) genotypes from clinical stool samples: polymerase chain reaction and direct molecular typing, culture characteristics, and serotyping.

Molecular (polymerase chain reaction [PCR]) methods are increasingly used to detect and type human enteroviruses (HEVs) and parechoviruses (HPeV). Here, we assessed their value in comparison to virus culture and serotyping for detection and typing of HEV and HPeV in stool samples from hospitalized patients. By use of real-time PCR, 221/1174 patients (18.8%) were found positive for HEV/HPeV. By cell culture, a virus could be isolated from 107 of the HEV/HPeV PCR-positive samples. Culture efficiency was correlated to the Ct value, (geno)type, and cell lines used. Of the HEV/HPeV PCR-positive samples, 47% could be genotyped by VP1 genotyping and 25% by serotyping. In conclusion, PCR detection of HEV/HPeV from stool is more sensitive than virus culture, particularly for coxsackieviruses A and HPeVs. However, the genotyping method used here could identify only 47% of the HEV/HPeV strains. Further optimization and validation of direct genotyping are needed, and clinical relevance of HEV/HPeV detection in stool needs to be determined.

Fourth human parechovirus serotype.

We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth HPeV serotype.