Davis Ferreira

Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase

Abstract Zika virus is an emerging mosquito-borne flavivirus currently causing large epidemics in the Pacific Ocean region and Brazil. Clinically, Zika fever resembles dengue fever, but is less severe. Whereas the clinical syndrome and laboratory diagnostic procedures have been described, little attention was paid to the immunology of the disease and its possible use for clinical follow-up of patients. Here, we investigate the role of cytokines in the pathogenesis of Zika fever in travelers returning from Asia, the Pacific, and Brazil. Polyfunctional T cell activation (Th1, Th2, Th9, and Th17 response) was seen during the acute phase characterized by respective cytokine level increases, followed by a decrease in the reconvalescent phase.

Prolonged detection of Zika virus RNA in urine samples during the ongoing Zika virus epidemic in Brazil.

Zika virus (ZIKV) is an emerging mosquito-borne virus that elongs to the genus Flavivirus. ZIKV may cause Zika virus disease ZVD) in humans that is characterized by fever, headache, myalgia nd rash. The first autochthonous transmission of ZIKV in Brazil was emonstrated in 2015 for patients from Rio Grande do Norte [1] and t was shown that the epidemic ZIKV strain in Brazil belong to the sian lineage [1,2]. Diagnosis of ZVD in humans is mainly based on NA detection in serum or plasma samples. Specific antibody detecion is mostly hampered due to strong serological cross-reactivity ith other circulating flaviviruses such as dengue virus or yellow ever virus [3–6]. Thus, there is an urgent need for a ZIKV diagostic protocol in Brazil that is effective in any ZIKV epidemic cenario, leading to a rapid, reliable and prolonged ZIKV RNA detecion. Paired samples of urine and serum from seven Brazilian ZVD atients were used for this study. For two additional ZVD patients nly urine samples were collected for a longer period after the onset f symptoms (ethical committee 80709). Viral RNA was extracted sing RTP® Pathogen Kit (Stratec, Birkenfeld—Germany), 400 l as used (for both, sera and urine samples), eluted in 50 l Elution uffer and ZIKV RT-PCR was performed according to Waehre et al. 7] using the Thermal Cycler peqLab—model peqstar 96X Univeral Gradient. Amplified fragments ∼200 pb were Sanger sequenced or confirmation. ZIKV RNA was detected in sera collected 2 days fter onset of symptoms, however urine samples from the same day ere tested negative. ZIKV RNA was first detectable in urine 4 days fter onset of symptoms, suggesting the beginning of renal excreion of ZIKV. For one patient ZIKV RNA excretion was observed until 4 days after onset of symptoms. In addition, our study demontrated higher levels of ZIKV RNA in urine when compared to serum. his was also observed by Gourinat et al. [8] during the ZVD epiemic in French Polynesia, reinforcing the evidence that the use f urine can increase the number of laboratory confirmed cases in n epidemic setting. The nucleotide sequences obtained with the mplicons confirmed the presence of the Asian lineage of ZIKV in io de Janeiro. In conclusion, urine samples should be considered s an important alternative to serum or plasma for the detection of IKV RNA because of a longer period of RNA detection, higher RNA evels, and less invasive sample collection.

Deletions in the Transmembrane Domain of a Sindbis Virus Glycoprotein Alter Virus Infectivity, Stability, and Host Range

ABSTRACT The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and protein-membrane interactions during virus assembly.

Synthesis and antiviral activity of new 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids derivatives

The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (3a-l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) derivatives was achieved with an efficient synthetic route. Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l) (52–82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c) (50–60%), respectively. Subsequent hydrolysis of the esters afforded the corresponding carboxylic acids (3a–l) (86–93%) and (5a–c) in high yield (80–93%). Inhibitory effects of 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids. Derivatives on Herpes simplex virus type 1 (HSV-1), Mayaro virus (MAY) and vesicular stomatitis virus (VSV) were investigated. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against HSV-1, MAY, and VSV virus with EC50 values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of these compounds showed toxicity for Vero cells.

Two lineages of dengue virus type 2, Brazil.

To the Editor: Dengue viruses (DENVs) belong to the genus Flavivirus (family Flaviviridae) and exist as 4 antigenic types, serotypes 1–4, each with well-defined genotypes. Dengue virus is associated with clinical manifestations that range from asymptomatic infections and relatively mild disease (classic dengue fever) to more severe forms of dengue hemorrhagic fever and dengue shock syndrome. Dengue has become one of the most serious vector-borne diseases in humans. The World Health Organization estimates that 2.5 billion persons live in dengue-endemic areas and >50 million are infected annually (1). In 1986, dengue virus type 1 (DENV-1) caused an outbreak in the state of Rio de Janeiro and has since become a public health concern and threat in Brazil. (2). In 1990, DENV-2 was reported in the state of Rio de Janeiro, where the first severe forms of dengue hemorrhagic fever and fatal cases of dengue shock syndrome were documented. The disease gradually spread to other regions of the country (3). In 2002, DENV-3 caused the most severe dengue outbreak in the country and sporadic outbreaks continued to be documented through 2005 (4). Since 1990, two additional epidemics caused by DENV-2 have occurred (1998 and 2007–2008) in Brazil. A severe DENV-2 epidemic in the state of Rio de Janeiro began in 2007 and continued in 2008; a total of 255,818 cases and 252 deaths were reported (5). This epidemic prompted us to investigate the genetic relatedness of DENV-2 for all of these epidemics. DENV-2 isolates from these epidemic periods were subjected to sequencing and comparison. Gross sequences of DENV-2 isolates from all epidemic periods grouped with sequences from DENV-2 American/Asian genotype; this finding was expected because this genotype is circulating in the Americas (6,7). Sequences of DENV-2 isolates from the 1998 epidemic grouped with sequences of DENV-2 isolates from the 1990 epidemic (data not shown) suggesting that viruses circulating during these 2 epidemic periods belong to the same lineage of the DENV-2 strain originally found in the state of Rio de Janeiro. However, sequences of DENV-2 isolates from 2007/2008 epidemics grouped separately and distinctly from the 1990 and 1998 DENV-2 isolates and represented a monophyletic group in the phylogenetic tree with bootstraps of 98% (Figure). This result shows a temporal circulation of genetically different viruses in Rio de Janeiro that could be a result of local evolution of DENV-2 since its introduction in 1990, or even an introduction of a new lineage of DENV-2 in the region. Figure Neighbor-joining phylogenetic tree of 68 complete envelope (E) gene sequences of dengue virus type 2 (DENV-2). Only bootstrap values >80% are shown. DENV-2 sequences obtained from 21 patients infected during the 1990, 1998, and 2007–2008 ... A study conducted by Aquino et al. (7) showed that Paraguayan DENV-2 strains could be grouped as 2 distinct variants within the American/Asian genotype, thus further supporting that the introduction of new DENV-2 variants may likely associate with the shift of dominant serotypes from DENV-3 to DENV-2 in 2005 and might have caused an outbreak of DENV-2. Our results are consistent with this scenario because was a shift of a dominant serotype from DENV-3 to DENV-2 that was observed in 2008 in Rio de Janeiro. However, other factors, such as immunity level to DENV-3 and DENV-2, could explain the shift of dominant serotype besides the circulation of a new viral variant. Because the dengue outbreaks of 2007 and 2008 were the most severe of the dengue infections in Brazil in terms of number of cases and deaths, this genetically distinct DENV-2 could have contributed to this pathogenic profile. Additionally, these samples came from disperse locations in Rio de Janeiro and we do not believe that there is a clustering issue in our sampling. However, again, other factors must be considered as contributors to this scenario because of the intrinsic properties of this distinct virus, host susceptibility, and secondary cases of infection. In addition, detailed examination of amino acid sequences of Brazilian DENV-2 strains isolated in 1998 and 2008 showed 6 aa substitutions in the envelope gene: V129I, L131Q, I170T, E203D, M340T, and I380V. Our results support the notion that aa positions at 129 and 131 in the envelope gene are critical genetic markers for phylogenetic classification of DENV-2 (7–9). Notably, residue 131 in the envelope gene is located within a pH-dependent hinge region at the interface between domains I and II of the envelope protein. Mutations at this region may affect the pH threshold of fusion and the process of conformational changes (10). Our results suggest the circulation of genetically different DENV-2 in Brazil and that these viruses may have a role in severity of dengue diseases. These findings can help to further understand the complex dynamic pathogenic profile of dengue viruses and their circulation in dengue-endemic regions.

Morphological variants of Sindbis virus produced by a mutation in the capsid protein.

Sindbis virus is a complex aggregate of RNA, protein and lipid. The virus is organized as two nested T = 4 icosahedral protein shells between which is sandwiched a lipid bilayer. The virus RNA resides within the inner protein shell. The inner protein shell is attached to the outer protein shell through contacts to proteins in the outer shell, which penetrate the lipid bilayer. The data presented in the following manuscript show that mutations in the capsid protein can result in the assembly of the virus structural proteins into icosahedra of different triangulation numbers. The triangulation numbers calculated, for these morphological variants, follow the sequence T = 4, 9, 16, 25 and 36. All fall into the class P = 1 of icosadeltahedra as was predicted by. The data support their hypothesis that families of icosahedra would be developed by altering the distance between the points of insertion of the five-fold axis. This capsid protein defect also results in the incorporation of much of the capsid protein, into large cytoplasmic aggregates of protein and RNA. These observations support models suggesting that the geometry of a pre-formed nucleocapsid organizes the assembly of the virus membrane proteins into a structure of identical configuration and argues against models suggesting that assembly of the membrane glycoproteins directs the assembly of the nucleocapsid.

Mutations in the Endodomain of Sindbis Virus Glycoprotein E2 Define Sequences Critical for Virus Assembly

ABSTRACT Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (Δ406-407, Δ409-411, and Δ414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.

Alphavirus genome delivery occurs directly at the plasma membrane in a time and temperature dependent process

ABSTRACT It is widely held that arboviruses such as the alphavirus Sindbis virus gain entry into cells by a process of receptor-mediated endocytosis followed by membrane fusion in the acid environment of the endosome. We have used an approach of direct observation of Sindbis virus entry into cells by electron microscopy and immunolabeling of virus proteins with antibodies conjugated to gold beads. We found that upon attaching to the cell surface, intact RNA-containing viruses became empty shells that could be identified only by antibody labeling. We found that the rate at which full particles were converted to empty particles increased with time and temperature. We found that this entry event takes place at temperatures that inhibit both endosome formation and membrane fusion. We conclude that entry of alphaviruses is by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins.

Single Amino Acid Insertions at the Junction of the Sindbis Virus E2 Transmembrane Domain and Endodomain Disrupt Virus Envelopment and Alter Infectivity

ABSTRACT The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 ΔK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 ΔK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K→F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.