Molecular detection and phylogenetic analysis of Cyprinid herpesvirus 3 in Brazilian ornamental fish.

Cyprinid herpesvirus 3 has a worldwide distribution and presents high mortality rates in species of Cyprinus carpio, causing serious economic loss to the global aquaculture industry. The description of this infection in other ornamental fish species is still limited. For this purpose, 100 ornamental fish from 24 different species were tested by PCR for Cyprinid hespesvirus 3 and the positive samples represented 6% of the tested samples. Phylogenetic reconstruction, based on the Thymidine Kinase gene, revealed the existence of two distinct clades. One clade grouped a Brazilian sample with European and Asian genotypes of CyHV-3 and a second clade, containing only Brazilian sequences described in this study. All of the Brazilian sequences showed identity values greater than 97.7% when compared to each other. This is the first report of the occurrence of Cyprinid herpesvirus 3 in ornamental fish species in Brazil. These results in association with further studies of viral isolation and characterization can help in establishing effective surveillance and disease control program.

Norovirus GII and astrovirus in shellfish from a mangrove region in Cananéia, Brazil: molecular detection and characterization.

In recent years, annual cases of gastroenteritis have been reported in the world at high rates, suggesting an association with the consumption of shellfish with enteric viruses in their tissues. Anthropic activities are considered a source of environmental pollution and the main responsible for contamination by pathogenic microorganisms in aquatic environments. The objective of this study was to evaluate, by RT-semi-nested PCR, the presence of astrovirus (AstV) and norovirus genogroup II (NoV GII) in mussels (Mytella falcata) and oysters (Crassostrea brasiliana) collected in two sites of the Lagunar Complex of Cananéia, State of São Paulo, Brazil. A total of 150 samples of mussels and oysters (75 samples each) were analyzed. AstV was not identified in any shellfish sample. NoV GII was detected in 21 samples (14%), 8 mussel samples (38%), and 13 oyster samples (62%). From the 21 positive samples, 16 were analyzed by nucleotide sequencing. The molecular characterization revealed that Brazilian samples were grouped into clades along with other sequences from Brazil, Japan, and Mexico. There was 93.8-100% amino acid sequence similarity among the samples in this study and > 94.9% when compared with the strains isolated from clinical cases in Brazil. The screening of shellfish for the presence of health-significant enteric viruses can help prevent outbreaks among consumers and contribute to the improvement of the estuarine environment.

Effects of triploid induction on innate immunity and hematology in Astyanax altiparanae.

Triploid induction is a promising biotechnique that could be used to enhance aquaculture yields in the near future. However, studies conducted with several fish species have demonstrated that the presence of an extra set of chromosomes may result in deleterious health effects. Furthermore, studies of fish immune responses still need to be conducted before these specimens can be readily commercialized. In the study presented herein, we evaluated the effects of triploid induction on hematology, erythrocyte morphometry and morphology, phagocytosis, and the expression levels of IL-1ß and TGF-ß using specimens of the Neotropical species, Astyanax altiparanae. In general, the cell counts of erythrocytes, leukocytes, and neutrophils in triploid fish were lower than those in diploid fish. The erythrocytes of triploid fish were larger than those found in diploid fish, but also demonstrated considerably higher frequencies of cellular and nuclear abnormalities. Although not statistically significant, triploid induction resulted in a phagocytic capacity (PC) 20% lower than that found with diploid fish. No notable differences were observed in phagocytic index (PI). Gene expression levels for the cytokine IL-1 were lower in tissues from the head kidney, liver, and spleen of triploid fish with respect to diploid fish. Gene expression levels of TGF-ß were lower only in the spleen of triploids compared to diploids. In conclusion, triploid induction resulted in A. altiparanae specimens with immune impairments and potentially lower resistances to disease and low-quality environments.

Genomic sequencing of a frog virus 3 strain from cultured American bullfrogs (Lithobates catesbeianus) in Brazil.

Frog virus 3 (FV3) was detected in cultured bullfrogs in Southeast Brazil. Phylodynamic analysis revealed recombination events in this strain that were nearly identical to those detected in North American and Brazilian FV3 strains. These data suggest that international trade of live bullfrogs has spread recombinant strains of FV3.

Detection and molecular characterization of Lymphocystivirus in Brazilian ornamental fish.

The aim of this study is to report the occurrence of Lymphocystivirus in Brazilian ornamental fish. From 25 ornamental fish species submitted for molecular diagnosis, only one sample (Pomacanthus imperator) was positive, and its viral nucleotide sequence obtained clustered with sequences of genotype VII. To our knowledge, this is the first report on the genetic characterization of Lymphocystivirus in Brazil.

Genetic diversity of bovine Picobirnavirus, Brazil.

Picobirnaviruses (PBVs) are emerging and opportunistic viruses with possible zoonotic potential. In this study, we present the detection, molecular characterization, and genotypic differentiation of PBVs from genogroup I in bovine stool samples from different Brazilian regions. A high proportion of PCR-positive samples (23.4%) was detected in a total of 77 analyzed. Nucleotide identity, alignment, and phylogenetic analyses revealed high diversity among the studied sequences. The results obtained indicate, for the first time, the circulation of bovine PBVs belonging to genogroup I in different Brazilian states, with heterogeneous phylogenetic-clustering profiles.

Molecular detection and phylogenetic analysis of megalocytivirus in Brazilian ornamental fish.

Megalocytiviruses have a worldwide distribution, causing serious economic loss to the global aquaculture industry. They also present a threat to ornamental fish trade because megalocytiviral infections have unspecified symptoms, making early diagnosis difficult. In this study, 100 ornamental fish from 24 different species were tested by PCR for megalocytivirus, with a 47% positive rate being identified. Phylogenetic reconstruction, based on the major capsid protein (MCP) gene, clustered all Brazilian samples into a single clade, showing identity values ranging from 99% to 100% when compared to each other. This is the first report of megalocytivirus infection in some ornamental fish species in Brazil.

Molecular characterization and genetic diversity of bovine Kobuvirus, Brazil.

It is suggested that Bovine kobuvirus (BKV) is involved in the etiology of gastroenteric diseases especially among calves; however, this association remains unknown. This study evaluated 216 fecal samples from cattle with and without diarrhea symptoms obtained from different regions of Brazil. A 216 bp fragment of the BKV 3D gene was amplified by RT-PCR in 14.4 % (31/216) of the studied samples, and 17 samples were subjected to nucleotide sequencing. All positive samples were obtained from animals aged less than 5 months, and most of animals presented diarrhea (p < 0.05). Phylogenetic analyses showed that the obtained sequences were grouped within the genogroup 2 of BKV forming subclades specific for each Brazilian municipality sampled. In addition, the alignment of the sequences revealed differences of nucleotides between sequences from different locations. Our results indicate for the first time that there is a regional genotypic differentiation of BKV in Brazil.

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil.

Bovine astrovirus (BoAstV) is associated with gastroenterical disorders such as diarrhea, particularly in neonates and immunocompromised animals. Its prevalence is >60 % in the first five weeks of the animal's life. The aim of this study was to detect and perform a phylogenetic analysis of BoAstV in Brazilian cattle. A prevalence of 14.3 % of BoAstV in fecal samples from 272 head of cattle from different Brazilian states was detected, and 11 samples were analyzed by nucleotide sequencing. The majority of positive samples were obtained from diarrheic animals (p < 0.01). Phylogenetic analysis revealed that Brazilian samples were grouped in clades along with other BoAstV isolates. There was 74.3 %-96.5 % amino acid sequence similarity between the samples in this study and >74.8 % when compared with reference samples for enteric BoAstV. Our results indicate, for the first time, the occurrence of BoAstV circulation in cattle from different regions of Brazil, prevalently in diarrheic calves.

Association of -308G/A polymorphism in the tumor necrosis factor-alpha gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeirão Preto region, Brazil.

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF -308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis.

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.

Natural host relationships and genetic diversity of rodent-associated hantaviruses in southeastern Brazil.

OBJECTIVE: Hantaviruses are rodent-borne RNA viruses that have caused hantavirus cardiopulmonary syndrome in several Brazilian regions. In the present study, geographical distribution, seroprevalence, natural host range, and phylogenetic relations of rodent-associated hantaviruses collected from seven counties of Southeastern Brazil were evaluated. METHODS: ELISA, RT-PCR and phylogenetic analysis were used in this study. RESULTS: Antibodies to hantavirus were detected in Bolomys lasiurus, Akodon sp. and Oligoryzomys sp., performing an overall seroprevalence of 5.17%. All seropositive rodents were associated with grasslands or woods surrounded by sugar cane fields. Phylogenetic analysis of partial S- and M-segment sequences showed that viral sequences isolated from B. lasiurus specimens clustered with Araraquara virus. However, a sequence from Akodon sp. shared 100% similarity with Argentinian/Chilean viruses based on the partial S-segment amino acid sequence. CONCLUSION: These results indicate that there are associations between rodent reservoirs and hantaviruses in some regions of Southeastern Brazil, and suggest the existence of additional hantavirus genetic diversity and host ecology in these areas.

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus.

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3h after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus.