Clinical and laboratory evidence of Haff disease - case series from an outbreak in Salvador, Brazil, December 2016 to April 2017.

We describe a series of 15 Haff disease cases from an outbreak in Salvador, Brazil, starting early December 2016. Eleven cases were grouped in four family clusters of two to four individuals, four were isolated cases. All but one patient consumed cooked fish; 11 within 24h before symptoms onset. Cases consumed 'Olho de Boi' (Seriola spp.) and 'Badejo' (Mycteroperca spp.). A total of 67 cases were detected, the last case was reported on 5 April 2017.

Distinct Zika Virus Lineage in Salvador, Bahia, Brazil.

Sequencing of isolates from patients in Bahia, Brazil, where most Zika virus cases in Brazil have been reported, resulted in 11 whole and partial Zika virus genomes. Phylogenetic analyses revealed a well-supported Bahia-specific Zika virus lineage, which indicates sustained Zika virus circulation in Salvador, Bahia's capital city, since mid-2014.

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing.

Metagenomic next-generation sequencing (mNGS) of samples from 15 patients with documented Zika virus (ZIKV) infection in Bahia, Brazil, from April 2015 to January 2016 identified coinfections with chikungunya virus (CHIKV) in 2 of 15 ZIKV-positive cases by PCR (13.3%). While generally nonspecific, the clinical presentation corresponding to these two CHIKV/ZIKV coinfections reflected infection by the virus present at a higher titer. Aside from CHIKV and ZIKV, coinfections of other viral pathogens were not detected. The mNGS approach is promising for differential diagnosis of acute febrile illness and identification of coinfections, although targeted arbovirus screening may be sufficient in the current ZIKV outbreak setting.

Ion torrent-based nasopharyngeal swab metatranscriptomics in COVID-19.

Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection of the virus.

Betulinic Acid Exerts Cytoprotective Activity on Zika Virus-Infected Neural Progenitor Cells.

Zika virus (ZIKV), a member of the Flaviviridae family, was brought into the spotlight due to its widespread and increased pathogenicity, including Guillain-Barré syndrome and microcephaly. Neural progenitor cells (NPCs), which are multipotent cells capable of differentiating into the major neural phenotypes, are very susceptible to ZIKV infection. Given the complications of ZIKV infection and potential harm to public health, effective treatment options are urgently needed. Betulinic acid (BA), an abundant terpenoid of the lupane group, displays several biological activities, including neuroprotective effects. Here we demonstrate that Sox2+ NPCs, which are highly susceptible to ZIKV when compared to their neuronal counterparts, are protected against ZIKV-induced cell death when treated with BA. Similarly, the population of Sox2+ and Casp3+ NPCs found in ZIKV-infected cerebral organoids was significantly higher in the presence of BA than in untreated controls. Moreover, well-preserved structures were found in BA-treated organoids in contrast to ZIKV-infected controls. Bioinformatics analysis indicated Akt pathway activation by BA treatment. This was confirmed by phosphorylated Akt analysis, both in BA-treated NPCs and brain organoids, as shown by immunoblotting and immunofluorescence analyses, respectively. Taken together, these data suggest a neuroprotective role of BA in ZIKV-infected NPCs.

Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection.

Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their long-term effects are not completely understood. The majority of the methods for ZIKV diagnosis are based on the detection of IgM antibodies, which are the first signs of immunological response. However, the detection of IgG antibodies can be an important approach for ZIKV past infection diagnosis, especially for pregnant women, helping the comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for efficient ZIKV or antibodies detection. Quantum dots (QD) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. Anti-IgG antibodies were successfully conjugated with QDs and applied in a fluorescence sensing nanoplatform. After optimization using IgG antibodies, the conjugates were employed to detect anti-ZIKV IgG antibodies in polystyrene microplates sensitized with ZIKV envelope E protein. The nanoplatform was able to detect anti-ZIKV IgG antibodies in a concentration at least 100-fold lower than the amount expected for protein E immune response. Moreover, conjugates were able to detect the antibodies for at least 4 months. Thus, our results showed that this QDs-based fluoroimmunoplatform can be considered practical, simple and promising to detect Zika past infections and/or monitoring immune response in vaccine trials.

N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection.

Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration.IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells.

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly.

Asymptomatic oral human papillomavirus (HPV) infection in women with a histopathologic diagnosis of genital HPV.

The oral route of human papillomavirus (HPV) transmission is not fully understood. It has been suggested that genital infection can act as a reservoir for oral HPV infection. We investigated the presence of oral HPV DNA and anti-HPV IgA in the buccal cavity of patients with a histopathologic diagnosis of cervical HPV infection. One hundred women underwent oral clinical examinations to detect HPV-DNA by polymerase chain reaction and salivary anti-HPV IgA by indirect immunofluorescence. Information on the personal habits of all the women was collected in personal interviews. Our results showed that 99% of the patients had no clinical manifestations of oral HPV. However, HPV DNA was detected in 81% of oral mucosa samples, and anti-HPV IgA was detected in the saliva of 44% of the patients. Consumption of alcoholic beverages was significantly associated with detection of oral HPV DNA and salivary anti-HPV IgA. Other behavioral risk factors associated with oral HPV and anti-HPV IgA are also discussed. In conclusion, patients with genital HPV infection are at risk for subclinical oral HPV infection. Thus, a molecular assay might be necessary to diagnose such infections.

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli.

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.