Global landscape of coxsackieviruses in human health.

Coxsackieviruses-induced infections, particularly in infants and young children, are one of the most important public health issues in low- and middle-income countries, where the surveillance system varies substantially, and these manifestations have been disregarded. They are widespread throughout the world and are responsible for a broad spectrum of human diseases, from mildly symptomatic conditions to severe acute and chronic disorders. Coxsackieviruses (CV) have been found to have 27 identified genotypes, with overlaps in clinical phenotypes between genotypes. In this review, we present a concise overview of the most recent studies and findings of coxsackieviruses-associated disorders, along with epidemiological data that provides comprehensive details on the distribution, variability, and clinical manifestations of different CV types. We also highlight the significant roles that CV infections play in the emergence of neurodegenerative illnesses and their effects on neurocognition. The current role of CVs in oncolytic virotherapy is also mentioned. This review provides readers with a better understanding of coxsackieviruses-associated disorders and pointing the impact that CV infections can have on different organs with variable pathogenicity. A deeper knowledge of these infections could have implications in designing current surveillance and prevention strategies related to severe CVs-caused infections, as well as encourage studies to identify the emergence of more pathogenic types and the etiology of the most common and most severe disorders associated with coxsackievirus infection.

Effectiveness of Household Disinfection Techniques to Remove SARS-CoV-2 from Cloth Masks.

To assess the efficacy of washing cloth masks, we simulated SARS-CoV-2 contamination in tricoline fabric and tested decontaminants to reduce viral particles. Viral suspensions using two variants (B.1.1.28 and P.1) were inoculated in these fabrics, and the inactivation kinetics were evaluated after washing with various household disinfection products (Soap powder, Lysoform®, Hypochlorite sodium and 70% Alcohol), rinse numbers, and exposure times. Afterward, the fabrics were washed in sterile water, and viral RNA was extracted and amplified using RT-qPCR. Finally, viral replication in cell cultures was examined. Our findings show that all biocidal treatments successfully disinfected the tissue tested. Some products showed less reduction in viral loads, such as soap powder (1.60 × 104, 1.04 × 103), soap powder and Lysoform® (1.60 × 104, 1.04 × 103), and alcohol 70% (1.02 × 103, 5.91 × 101), respectively. However, when sodium hypochlorite was used, this reduction was significantly increased (viral inactivation in 100% of the washes). After the first wash, the reduction in the number of viral particles was greater for the P.1 variant than for the B.1.1.28 variant (W = 51,759, p < 0.05). In conclusion, the role of sodium hypochlorite in cloth mask disinfection may also have implications for future health emergencies as well as recommendation by WHO.

Analysis of Coxsackievirus B5 Infections in the Central Nervous System in Brazil: Insights into Molecular Epidemiology and Genetic Diversity.

Coxsackievirus B5 (CVB5) is one of the most prevalent enteroviruses types in humans and causes annual epidemics worldwide. In the present study, we explored viral genetic diversity, molecular and epidemiological aspects of CVB5 obtained from cerebrospinal fluid and stool samples of patients with aseptic meningitis or acute flaccid paralysis, information that is still scarce in Brazil. From 2005 to 2018, 57 isolates of CVB5 were identified in the scope of the Brazilian Poliomyelitis Surveillance Program. Phylogenetic analyses of VP1 sequences revealed the circulation of two CVB5 genogroups, with genogroup B circulating until 2017, further replaced by genogroup A. Network analysis based on deduced amino acid sequences showed important substitutions in residues known to play critical roles in viral host tropism, cell entry, and viral antigenicity. Amino acid substitutions were investigated by the Protein Variation Effect Analyzer (PROVEAN) tool, which revealed two deleterious substitutions: T130N and T130A. To the best of our knowledge, this is the first report to use in silico approaches to determine the putative impact of amino acid substitutions on the CVB5 capsid structure. This work provides valuable information on CVB5 diversity associated with central nervous system (CNS) infections, highlighting the importance of evaluating the biological impact of certain amino acids substitutions associated with epidemiological and structural analyses.

Viral and Prion Infections Associated with Central Nervous System Syndromes in Brazil.

Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.

Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005-2017).

Due to the advanced stage of polio eradication, the possible role of non-polio enteroviruses (NPEVs) associated to acute flaccid paralysis (AFP) cases has been highlighted. In this study, we described epidemiological aspects of NPEVs infections associated to AFP and explore the viral genetic diversity, information still scarce in Brazil. From 2005 to 2017, 6707 stool samples were collected in the scope of the Brazilian Poliomyelitis Surveillance Program. NPEVs were isolated in 359 samples (5.3%) and 341 (94.9%) were genotyped. About 46 different NPEV types were identified with the following detection pattern EV-B > EV-A > EV-C. The major EV-types were CVA2, CV4, EV-A71, CVB3, CVB5, E6, E7, E11, CVA13 and EV-C99, which corresponds to 51.6% of the total. Uncommon types, such as CVA12, EV-90 and CVA11, were also identified. Different E6 genogroups were observed, prevailing the GenIII, despite periods of co-circulation, and replacement of genogroups along time. CVA2 sequences were classified as genotype C and data suggested its dispersion in South-American countries. CVA13 viruses belonged to cluster B and Venezuelan viruses composed a new putative cluster. This study provides extensive information on enterovirus diversity associated with AFP, reinforcing the need of tailoring current surveillance strategies to timely monitor emergence/re-emergence of NPEVs.

Current Understanding of the Role of Cholesterol in the Life Cycle of Alphaviruses.

Enveloped viruses rely on different lipid classes present in cell membranes to accomplish several steps of their life cycle in the host. Particularly for alphaviruses, a medically important group of arboviruses, which are part of the Togaviridae family, cholesterol seems to be a critical lipid exploited during infection, although its relevance may vary depending on which stage of the virus life cycle is under consideration and whether infection takes place in vertebrate or invertebrate hosts. In this review, the role of cholesterol in both early and late events of alphavirus infection and how viral replication may affect cholesterol metabolism are summarized, taking into account studies on Old World and New World alphaviruses in different cell lines. Moreover, the importance of cholesterol for the structural stability of alphavirus particles is also discussed, shedding light on the role played by this lipid when they leave the host cell.

Re-emergence of a coxsackievirus A24 variant causing acute hemorrhagic conjunctivitis in Brazil from 2017 to 2018.

A large outbreak (over 200,000 cases) of acute hemorrhagic conjunctivitis (AHC) took place in Brazil during the summer of 2017/2018, seven years after a nationwide epidemic, which occurred in 2011. To identify the etiological agent, 80 conjunctival swabs from patients with a clinical presentation suggestive of AHC were analyzed at the national enterovirus laboratory. Real-time RT-PCR for human enteroviruses was performed, and enterovirus RNA was detected in 91.25% (73/80) of the specimens. Twenty-nine swab fluids were used to inoculate cell cultures (RD and Hep2C), and 72.4% (21/29) yielded a cytopathic effect. Genotype IV coxsackievirus A24v (CV-A24v) was identified as the causative agent of the outbreak. Phylogenetic analysis based on the VP1 gene revealed that Brazilian isolates were genetically related to strains that caused an outbreak in French Guiana in 2017. Our results show the re-emergence of CV-A24v causing AHC outbreaks in Brazil between the end of 2017 and the beginning of 2018.

Enterovirus B74 associated with hand, foot and mouth disease.

Enterovirus 74 (EV-B74) has been associated with cases of acute flaccid paralysis (AFP) but it is not a commonly found enterovirus. In this work, we present the characterization of an EV-B74 detected from the serum sample of a one-year-old boy presenting with signs and symptoms clinically compatible with hand, foot and mouth disease (HFMD). This is the first report of EV-B74 in Brazil.

Fusion of a New World Alphavirus with Membrane Microdomains Involving Partially Reversible Conformational Changes in the Viral Spike Proteins.

Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry mechanism. In this study, we show for the first time the interaction of an enveloped virus with membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon acidification, although its kinetics was quite slow when compared to that of fusion with artificial liposomes demonstrated in a previous work. Surprisingly, when virus was previously exposed to acidic pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well as infectivity, and then reneutralized, its ability to fuse with membrane microdomains at low pH was retained. Interestingly, this observation correlated with a partial reversion of low pH-induced conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our results suggest that MAYV entry into host cells could alternatively involve internalization via lipid rafts and that the conformational changes triggered by low pH in the viral spike proteins during the entry process are partially reversible.

Acute flaccid paralysis laboratorial surveillance in a polio-free country: Brazil, 2005-2014.

The last case of paralytic poliomyelitis caused by wild poliovirus in Brazil occurred in 1989. The interruption of the indigenous poliovirus transmission was obtained through mass immunization campaigns to eligible children and an active epidemiological and laboratorial surveillance of all cases of acute flaccid paralysis (AFP) among children under 15 y of age. This paper describes and evaluates the performance of the AFP surveillance system in different geographic areas of Brazil between 2005 and 2014, using indicators recommended by WHO. AFP surveillance indicators as well as virological investigation of polio and non-polio enteroviruses in stool samples received in the laboratory were assessed from 2005-2014. During the period, 5463 cases of AFP were investigated. Of these, 55% were males and 45% were females. Those under 5 y of age represented 48% of all cases reported and investigated. AFP notification rate was within the acceptable values with mean value of 1.3 (North), 1.4 (Northeast), 1.1 (Southern), 1.0 (Southeast) and 1.4 (Midwest) cases of AFP per 100.000 population aged 15 y as well as the adequacy of fecal specimens received in the laboratory. Sabin- related polioviruses accounted for 1.7% of the isolates while, 6.7% were non-polio enterovirus with the values ranging from 5.0% to 8.9 %. No wild-type polio was found. The AFP epidemiological and laboratorial surveillance activities have been kept at appropriate levels in Brazil. These data are a very strong indication, which supports the status of country free of polio.

Inhibition of Mayaro virus infection by bovine lactoferrin.

Mayaro virus (MAYV) is an arbovirus linked to several sporadic outbreaks of a highly debilitating febrile illness in many regions of South America. MAYV is on the verge of urbanization from the Amazon region and no effective antiviral intervention is available against human infections. Our aim was to investigate whether bovine lactoferrin (bLf), an iron-binding glycoprotein, could hinder MAYV infection. We show that bLf promotes a strong inhibition of virus infection with no cytotoxic effects. Monitoring the effect of bLf on different stages of infection, we observed that virus entry into the cell is the heavily compromised event. Moreover, we found that binding of bLf to the cell is highly dependent on the sulfation of glycosaminoglycans, suggesting that bLf impairs virus entry by blocking these molecules. Our findings highlight the antiviral potential of bLf and reveal an effective strategy against one of the major emerging human pathogens in the neotropics.

The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

Envelope lipid-packing as a critical factor for the biological activity and stability of alphavirus particles isolated from mammalian and mosquito cells.

Alphaviruses are enveloped arboviruses. The viral envelope is derived from the host cell and is positioned between two icosahedral protein shells (T = 4). Because the viral envelope contains glycoproteins involved in cell recognition and entry, the integrity of the envelope is critical for the success of the early events of infection. Differing levels of cholesterol in different hosts leads to the production of alphaviruses with distinct levels of this sterol loaded in the envelope. Using Mayaro virus, a New World alphavirus, we investigated the role of cholesterol on the envelope of alphavirus particles assembled in either mammalian or mosquito cells. Our results show that although quite different in their cholesterol content, Mayaro virus particles obtained from both cells share a similar high level of lateral organization in their envelopes. This organization, as well as viral stability and infectivity, is severely compromised when cholesterol is depleted from the envelope of virus particles isolated from mammalian cells, but virus particles isolated from mosquito cells are relatively unaffected by cholesterol depletion. We suggest that it is not cholesterol itself, but rather the organization of the viral envelope, that is critical for the biological activity of alphaviruses.