Kudoa rousseauxii n. sp. (Cnidaria: Multivalvulida) Infects the Skeletal Muscles of the Freshwater Fish Brachyplatystoma rousseauxii in the Amazon River.

PURPOSE: Members of the genus Kudoa Meglitsch, 1947 are known to infect the muscles of commercially important fishes worldwide, including those in the order Siluriformes. This paper describes the occurrence of a new species of Kudoa in the catfish Brachyplatystoma rousseauxii based on morphological study and molecular analysis of the ribosomal RNA gene (SSU rDNA). METHODS: Fifteen specimens of Brachyplatystoma rousseauxii were purchased from fishing zones near Mosqueiro Island, Belém, Pará, Brazil. After necropsy, tissue samples and cysts were analyzed using a stereomicroscope, and fresh slides were viewed under a light microscope to confirm parasitic infection. The tissue fragments were removed and processed for molecular and histological analyses. RESULTS: Microscopic pseudocysts were found in the epaxial region of skeletal muscle fibers in 80% of the analyzed specimens. The myxospores were quadrangular with four shell valves (SV), pyriform polar capsules (PC), and internal symmetry. Phylogenetic analyses indicated that the new species formed a cluster with the species previously described in the Amazon, being close to two freshwater species. CONCLUSIONS: Morphological differences and molecular data of SSU rDNA support that Kudoa rousseauxii n. sp. is a new species that infects B. rousseauxii, a freshwater fish with intense migratory cycles that is widely captured and consumed in the Amazon.

In silico improvement of the cyanobacterial lectin microvirin and mannose interaction.

Lectins that bind to HIV envelope glycoprotein can inhibit virus-cell fusion and be used for rational drug design. This paper presents the results of an in silico approach to improve affinity interaction between the cyanobacterial lectin microvirin and its ligand Manα(1-2)Man. Comparative modeling and molecular dynamics tools were used. Additionally, the alanine scanning webserver was used to study the importance of protein residues in the binding site and to guide mutant production. The model obtained presented two homologous domains designated as domains A and B, each consisting of a single strand with triple and antiparallel ß-sheets of (ß1-ß3 and ß6-ß8). Disulfide bonds between the cysteines (Cys60-Cys80, Cys63-Cys78 and Cys8-Cys24) were also found. The highly conserved binding site, including residues Asn44, Ile45, Asp46, Gln54, Asn55, Glu58, Thr59, Gln81, Thr82 and Met83. The RMSD values of the di-mannose and the interaction site were very stable during the molecular dynamics. Calculations of the occupation time of the hydrogen bonds were made for the residues that showed interaction in the complex lectin and ligand. The residue that contributed most to the interaction with Manα(1-2)Man was Asn55. After validation, the model generated remained stable during the entire simulation. Despite its structural similarity with the template we used, our mutant (Thr82Arg) showed a higher affinity interaction with Manα(1-2)Man. Communicated by Ramaswamy H. Sarma.

Incidence of viral hepatitis in Brazil from 2009 to 2018: an epidemiological study of confirmed cases of viral hepatitis

Abstract INTRODUCTION: Viral hepatitis is a major public health problem. It is necessary to understand the epidemic, verifying the combination of biological and demographic characteristics. METHODS: This is an analytical ecological and epidemiological study. Confirmed case data from the Notification Disease Information System (SINAN) were used. RESULTS: From 2009-2018, SINAN confirmed 404,003 viral hepatitis cases in Brazil, with 12.49%, 37.06%, and 48.28% cases of hepatitis A, B, and C, respectively. CONCLUSIONS: In Brazil, 4,296 deaths were associated with viral hepatitis, of which 36.66% were associated with acute hepatitis B. The proportional distribution of cases varied among the five Brazilian regions.

Myxobolus freitasi n. sp. (Myxozoa: Bivalvulida), a parasite of the brain of the electric knifefish in the Brazilian Amazon region / Myxobolus freitasi n. sp. (Myxozoa: Bivalvulida), parasita do cérebro do peixe-faca elétrico na Amazônia brasileira

Abstract A total of 30 specimens of the Amazonian electric knifefish, Brachyhypopomus beebei Schultz, 1944 (Gymnotiformes: Hypopomidae), were collected from the Peixe-Boi River in the state of Pará, Brazil (1°06'59" S; 47°18'26" W). Fragments of the brain tissue were extracted for analysis via optical microscopy, and 18 specimens (60%) presented microparasites of the genus Myxobolus, with unequal capsules. The spores were 18.6 µm (17.7-19.8 µm) long and 8.6 µm (8.4-9.0 µm) wide; the largest polar capsule was 13.0 µm (12.4-13.4 µm) long and 5.6 µm (5.3-6.0 µm) wide, and the smallest capsule was 5.0 µm (4.5-5.3 µm) long and 2.5 µm (2.3-2.6 µm) wide. Infected brain fragments were extracted for histological processing and staining with hematoxylin-eosin and Ziehl-Neelsen. Some fragments were conserved in ethanol for molecular genetics analysis. A partial sequence of the 18S DNA gene was obtained from the spores, which did not correspond to any other sequences deposited in GenBank, although it did form a clade with other Myxobolus parasites of the nervous system. The morphological data, together with molecular phylogeny, supported the designation of a new species Myxobolus freitasi n. sp.

Resumo Um total de 30 espécimes do peixe-faca elétrico da Amazônia, Brachyhypopomus beebei Schultz, 1944 (Gymnotiformes: Hypopomidae), foram coletados no rio Peixe-Mani, no estado do Pará, Brasil (1 ° 06'59 "S; 47 ° 18 ' 26 "W). Fragmentos de tecido cerebral foram extraídos para análise em microscopia óptica, sendo que 18 espécimes (60%) apresentavam microparasitos do gênero Myxobolus, com cápsulas desiguais. Os esporos apresentavam 18,6 µm (17,7-19,8 µm) de comprimento e 8,6 µm (8,4-9,0 µm) de largura; a maior cápsula polar tinha 13,0 µm (12,4-13,4 µm) de comprimento e 5,6 µm (5,3-6,0 µm) de largura, e a menor cápsula tinha 5,0 µm (4,5-5,3 µm) de comprimento e 2,5 µm (2,3-2,6 µm) de largura. Fragmentos cerebrais infectados foram extraídos para processamento histológico e coloração com hematoxilina-eosina e Ziehl-Neelsen. Alguns fragmentos foram conservados em etanol para análise genética molecular. Dos esporos, foi obtida uma sequência parcial do gene 18S do DNA, que não correspondeu a nenhuma outra sequência depositada no GenBank, embora tenha formado um clado com outros parasitas do gênero Myxobolus do sistema nervoso. Os dados morfológicos, juntamente com a filogenia molecular, apoiaram a designação de uma nova espécie Myxobolus freitasi n. sp.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.

Anti-dengue virus activity of scytovirin and evaluation of point mutation effects by molecular dynamics and binding free energy calculations.

The absence of a specific treatment against DENV has led to intensive research into developing strategies for curing the infection. One lectin with high antiviral activity is scytovirin, which was isolated from the cyanobacterium Scytonema varium and has proven activity against HIV and Zaire Ebola Virus. To achieve the results presented here, we tested the affinity of full-length scytovirin, SD1 and SD2 separately, and six SD1 mutants for DENV glycoprotein E carbohydrate by Molecular Dynamics (MD) simulations and binding free energy calculations. It was possible to identify the key residues for protein-ligand interaction such as Glu10, Ala11, Pro17, Ans18, Arg30, Thr41, Ser42 and Arg43, which also has importance action against HIV. All binding free energy calculations showed negative values to ΔGbind of protein-DENV carbohydrate complexation. Additionally, these results are similar to the values of scytovirin and HIV gp120 carbohydrate complexation (-32.20 kcal/mol). Furthermore, we found that SD1 individually has more affinity to the carbohydrate and the Asn9, Glu10, Asn18, Arg30 and Arg43 demonstrated an important role in this matter. We also found that mutant G48R has better affinity (-34.10 kcal/mol) for the DENV carbohydrate than the wild type protein (-27.15 kcal/mol).

Henneguya paraensis n. sp. (Myxozoa; Myxosporea), a new gill parasite of the Amazonian fish Cichla temensis (Teleostei: Cichlidae): morphological and molecular aspects.

The present study describes light microscopy, transmission electron microscopy, and molecular analyses of a myxosporid found parasitizing the gill region of the teleost fish Cichla temensis, collected from the Tocantins River, near Cametá, Pará State, Brazil. The prevalence of infection was 60 %. The spore-containing cysts that were located in the gill lamellae were oval and whitish. The spores had a mean length of 42.3 ± 0.65 µm; fusiform body, 12.8 ± 0.42-µm long and 8.6 ± 0.32-µm wide; each of the two valves exhibited a tapering tail of 29.5 ± 0.73 µm length. The spores had two polar capsules, 7.4 ± 0.16-µm long by 2.6 ± 0.08-µm wide, containing a polar filament with 5-7 twists. The spores differ from the species previously described, and phylogenetic analysis based on spore morphology and molecular aspects indicated that the fish parasite Henneguya sp. has a strong trend to form clades mainly based on the environment and host order/family. Thus, we conclude that the species belongs to the family Myxobolidae, genus Henneguya, which comprises a new species: Henneguya paraensis n. sp.

Optimization of a molecular method for the diagnosis of canine babesiosis / Otimização de método molecular para diagnóstico da babesiose canina

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.

A babesiose é uma doença hemolítica de ocorrência mundial, causada por protozoários do gênero Babesia (Apicomplexa), que são transmitidos por carrapatos a diversos mamíferos, incluindo o homem. O objetivo deste estudo foi otimizar um método molecular para a detecção de fragmento do 18S rDNA de Babesia canis, Babesia vogeli, Babesia rossi ou Babesia gibsoni com base em uma única semi-nested (snPCR), comparando sua eficiência com um protocolo de PCR simples. Para isso, 100 amostras de sangue de cães com suspeita de hemoparasitoses foram analisadas e, enquanto o protocolo de PCR simples indicou somente 5% (5/100) de amostras positivas, o protocolo de snPCR, com 22% (22/100) de amostras positivas, apresentou maior sensibilidade (p valor = 0,0000). Este resultado está de acordo com outros estudos que mostram a maior sensibilidade de detecção dos testes baseado em nested ou snPCR. Assim, como uma forma de prevenir resultados falso-negativos devido à baixa parasitemia, sugere-se que este protocolo seja preferencialmente usado nos estudos epidemiológicos de babesiose canina, em especial naqueles que tratam da sua prevalência.

Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae)

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.