Genetic differentiation of Megalocytivirus by real time PCR and sequencing.

BACKGROUND: Megalocytiviruses (MCV) are double-stranded DNA viruses that infect fish. Two species within the genus are epidemiologically important for fish farming: red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV). The objective of this work was to study regions that allow the differentiation and correct diagnosis of RSIV and ISKNV. METHODS: The regions ORF450L, ORF342L, ORF077, and the intergenic region between ORF37 and ORF42R were sequenced and compared with samples from the database. RESULTS: The tree constructed using the sequencing of the PCR product Megalocytivirus. ORF077 separated the three major clades of MCV. RISV genotypes were well divided, but not ISKNV. All qPCRs tests showed acceptable repeatability values, that is, less than 5%. CONCLUSION: Two qPCRs for ISKNV detection and two for RSIV were considered suitable for use in the diagnosis and typing of MCV. The results of this study demonstrate the importance of an accurate evaluation of methodologies for the differentiation of MCV.

Detection of megalocytivirus in Oreochromis niloticus and Pseudoplatystoma corruscans in Brazil.

The infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus (MCV), a group of double-stranded DNA genome viruses. The aim of this study was to retrospectively analyze samples from suspected foci of MCV infection in freshwater fish in Brazil. Samples were collected from infected fish between 2017 and 2021. Phylogenetic analysis revealed 2 groups of MCV circulating in the country. A genetically homogeneous group formed a clade with ISKNV samples from different parts of the world. Only 2 of the sequences from the state of Goiás showed a small genetic distance when compared to the larger group in the same clade. This study describes the validation of 3 qPCR methods and the presence of MCV in Brazil since 2017, including a genotype not previously described.

First report of infectious spleen and kidney necrosis virus in Nile tilapia in Brazil.

In June 2020, an atypical fatal outbreak in a Brazilian Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. Phylogenetic analysis indicated that the virus belongs to the Clade 1 of ISKNV. This viral pathogen is associated with high mortality in the early stages of cultured Nile tilapia in the United States, Thailand and Ghana; however, until now, there have been no reports from ISKNV affecting cultured fish in Brazil. Additionally, in 14 out of 23 sampled fish, Streptococcus agalactiae, Edwardsiella tarda or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms and represents a new challenge to the aquaculture sector in the country.

Molecular detection of vector-borne agents in ectoparasites and reptiles from Brazil.

Trombidiformes and Mesostigmata mites, as well as Ixodida ticks, infest ectothermic tetrapods worldwide, potentially acting as vectors of bacteria, viruses and protozoa. The relationship among ectoparasites, transmitted pathogenic agents (e.g., Borrelia spp., Coxiella spp., Hepatozoon spp., and Rickettsia spp.) and ectothermic hosts has been scarcely investigated. This research focuses on a large collection of Brazilian herpetofauna screened for the presence of arthropod ectoparasites and vector-borne microbial agents. Reptiles (n = 121) and amphibians (n = 49) from various locations were infested by ectoparasites. Following genomic extraction, microbial agents were detected in 81 % of the Acari (i.e. n = 113 mites and n = 26 ticks). None of the mites, ticks and tissues from amphibians yielded positive results for any of the screened agents. Blood was collected from reptiles and processed through blood cytology and molecular analyses (n = 48). Of those, six snakes (12.5 %) showed intraerythrocytic alterations compatible with Hepatozoon spp. gamonts and Iridovirus inclusions. Hepatozoon spp. similar to Hepatozoon ayorgbor and Hepatozoon musa were molecularly identified from seven hosts, two mite and two tick species. Rickettsia spp. (e.g., Rickettsia amblyommatis, Rickettsia bellii-like, Rickettsia sp.) were detected molecularly from four mite species and Amblyomma rotundatum ticks. Phylogenetic analyses confirmed the molecular identification of the above-mentioned microbial agents of mites and ticks related to snakes and lizards. Overall, our findings highlighted that the Brazilian herpetofauna and its ectoparasites harbour potentially pathogenic agents, particularly from the northern and south-eastern regions. The detection of several species of spotted fever group Rickettsia pointed out the potential role of ectothermic hosts and related arthropod ectoparasites in the epidemiological cycle of these bacteria in Brazil.

Complete genome sequence and analysis of a novel lymphocystivirus detected in whitemouth croaker (Micropogonias furnieri): lymphocystis disease virus 4.

A novel lymphocystivirus causing typical signs of lymphocystis virus disease in whitemouth croaker (Micropogonias furnieri) on the coast of Uruguay was detected and described recently. Based on genetic analysis of some partially sequenced core genes, the virus seemed to differ from previously described members of the genus Lymphocystivirus. In this study, using next-generation sequencing, the whole genome of this virus was sequenced and analysed. The complete genome was found to be 211,086 bp in size, containing 148 predicted protein-coding regions, including the 26 core genes that seem to have a homologue in every iridovirus genome sequenced to date. Considering the current species demarcation criteria for the family Iridoviridae (genome organization, G+C content, amino acid sequence similarity, and phylogenetic relatedness of the core genes), the establishment of a novel species ("Lymphocystis disease virus 4") in the genus Lymphocystivirus is suggested.

Mosquito Iridescent Virus: New Records from Nature and Infections Using Strelkovimermis spiculatus (Mermithidae) as a Vector Under Laboratory Conditions.

Iridoviridae is a DNA virus family that affects both vertebrates and invertebrates. Immature aquatic stages of many dipteran species infected with iridovirus have been found in different places worldwide. The most represented genera of the Culicidae family are Aedes and Psorophora. To date, sixteen species of Aedes naturally infected with iridoviruses have been reported. Moreover, there are four records for the genus Psorophora, one for Culiseta, and two for Culex. In this paper, we report two new mosquito species as natural hosts of iridoviridae in Argentina: Aedes albifasciatus (Macquart) and Culex dolosus (Lynch Arribalzaga). We also analyzed the ability of a Cx. pipiens-Invertebrate Iridescent Virus to replicate in vivo in the larval stage of two mosquito species, Culex apicinus Philippi and Ae. aegypti (L.) using Strelkovimermis spiculatus as a vector, under laboratory conditions. Although Ae. aegypti is the most recognized mosquito vector of important arboviruses responsible for emergent diseases, Cx. apicinus and Ae. albifasciatus may also be implicated in enzootic or epizootic cycles of virus transmission, such as the St. Louis Encephalitis virus and the Western Equine Encephalomyelitis virus.

Diagnosis of lymphocystis disease in a novel host, the whitemouth croaker Micropogonias furnieri, associated with a putatively novel Lymphocystivirus species (LCDV-WC).

In 2015, an episode of lymphocystis disease (LCD) was detected in wild and cultured populations of whitemouth croaker Micropogonias furnieri off the coast of Uruguay. Fish of both origins were collected for histopathological and molecular investigations. Macroscopically, multinodular tumorlike masses were observed in the skin. Histological examination of these masses revealed enlarged cells with a hyaline capsule and basophilic inclusion bodies in the cytoplasm. The inclusion bodies were further examined by electron microscopy and showed icosahedral virions with a median diameter of 182 nm. Routine molecular investigations targeting the DNA polymerase and major capsid protein genes showed the presence of the DNA of an unknown lymphocystis disease virus (LCDV) in all specimens showing external signs of LCD. Subsequently, 4 other core genes were amplified and sequenced from the viral genome. Phylogenetic tree reconstruction based on the concatenated sequence of 6 core genes indicated that the virus undoubtedly belongs to the genus Lymphocystivirus. However, the core gene sequences of the whitemouth croaker LCDV differ markedly from those of the 3 known LCDVs, putatively representing a fourth LCDV species.

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.

Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America.

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.

A Preliminary Study of Largemouth Bass Virus in Mexico.

Disease outbreaks and mortalities caused by largemouth bass virus (LMBV) in largemouth bass ( Micropterus salmoides) have been reported in the US. Blood and mucus samples tested by PCR to assess the presence of LMBV in largemouth bass in northeastern Mexico were negative, and further monitoring is needed.

Red seabream iridovirus associated with cultured Florida pompano Trachinotus carolinus mortality in Central America.

Mariculture of Florida pompano Trachinotus carolinus in Central America has increased over the last few decades and it is now a highly valued food fish. High feed costs and infectious diseases are significant impediments to the expansion of mariculture. Members of the genus Megalocytivirus (MCV), subfamily Alphairidovirinae, within the family Iridoviridae, are emerging pathogens that negatively impact Asian mariculture. A significant mortality event in Florida pompano fingerlings cultured in Central America occurred in October 2014. Affected fish presented with abdominal distension, darkening of the skin, and periocular hemorrhages. Microscopic lesions included cytomegalic 'inclusion body-bearing cells' characterized by basophilic granular cytoplasmic inclusions in multiple organs. Transmission electron microscopy revealed arrays of hexagonal virions (155-180 nm in diameter) with electron-dense cores within the cytoplasm of cytomegalic cells. Pathological findings were suggestive of an MCV infection, and the diagnosis was later confirmed by partial PCR amplification and sequencing of the viral gene encoding the myristylated membrane protein. The viral sequence revealed that the fingerlings were infected with an MCV genotype, red seabream iridovirus (RSIV), previously reported only from epizootics in Asian mariculture. This case underscores the threat RSIV poses to global mariculture, including the production of Florida pompano in Central America.

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.

First outbreak of an infection with infectious spleen and kidney necrosis virus (ISKNV) in ornamental fish in Germany.

In 2014, infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus, was detected for the first time in ornamental fish in Germany. Since 2013, angelfish Pterophyllum spp. originating from Colombia have experienced significant epizootics in a number of German retailers' facilities. The diseased fish showed symptoms such as increased ventilation, swollen gills, and ulcerations of the skin. In 2014, diseased angelfish P. altum and platys Xiphophorus maculatus maintained in the same recirculating system were examined. Histopathological lesions included hypertrophic cells, single-cell necrosis, and an inflammatory infiltration of granulocytes, lymphocytes, and macrophages in liver, spleen, and kidney. Transmission electron microscopy revealed the presence of numerous polygonal viral particles (150 nm in diameter) within the cytoplasm of enlarged cells. A PCR assay for the detection of megalocytiviruses amplified 777 bp of major capsid protein gene that was 100% identical to ISKNV. This is the first report of an ISKNV outbreak in Germany that most probably was introduced by infected angelfish from Colombia. To our knowledge, this is the first report of ISKNV detected in fish imported from South America. Given the lethal nature of megalocytiviruses, proper biosecurity would seem prudent in countries like Germany where these emerging pathogens are not established.

Astaxanthin prevents in vitro auto-oxidative injury in human lymphocytes.

Upon mitogen sensitization, lymphocytes undergo proliferation by oxyradical-based mechanisms. Through continuous resting-restimulation cycles, lymphocytes accumulate auto-induced oxidative lesions which lead to cell dysfunction and limit their viability. Astaxanthin (ASTA) is a nutritional carotenoid that shows notable antioxidant properties. This study aims to evaluate whether the in vitro ASTA treatment can limit oxyradical production and auto-oxidative injury in human lymphocytes. Activated lymphocytes treated with 5 microM ASTA showed immediate lower rates of O(2)(*-) /H(2)O(2) production whilst NO* and intracellular Ca(2+) levels were concomitantly enhanced (24 h), the cytotoxicity test for ASTA showed a sigmoidal dose-response curve (LC50 = 11.67 +/- 0.42 microM), whereas higher activities of superoxide dismutase and catalase in 5 microM ASTA-treated lymphocytes were associated to significant lower indexes of oxidative injury. On the other hand, lower proliferative scores of ASTA lymphocytes might be a result of diminished intracellular levels of pivotal redox signaling molecules, such as H(2)O(2). Further studies are necessary to establish the ASTA-dose compensation point between minimizing oxidative damages and allowing efficient redox-mediated immune functions, such as proliferation, adhesion, and oxidative burst.

Mutations in the hydrophobic core and in the protein-RNA interface affect the packing and stability of icosahedral viruses.

The information required for successful assembly of an icosahedral virus is encoded in the native conformation of the capsid protein and in its interaction with the nucleic acid. Here we investigated how the packing and stability of virus capsids are sensitive to single amino acid substitutions in the coat protein. Tryptophan fluorescence, bis-8-anilinonaphthalene-1-sulfonate fluorescence, CD and light scattering were employed to measure urea- and pressure-induced effects on MS2 bacteriophage and temperature sensitive mutants. M88V and T45S particles were less stable than the wild-type forms and completely dissociated at 3.0 kbar of pressure. M88V and T45S mutants also had lower stability in the presence of urea. We propose that the lower stability of M88V particles is related to an increase in the cavity of the hydrophobic core. Bis-8-anilinonaphthalene-1-sulfonate fluorescence increased for the pressure-dissociated mutants but not for the urea-denatured samples, indicating that the final products were different. To verify reassembly of the particles, gel filtration chromatography and infectivity assays were performed. The phage titer was reduced dramatically when particles were treated with a high concentration of urea. In contrast, the phage titer recovered after high-pressure treatment. Thus, after pressure-induced dissociation of the virus, information for correct reassembly was preserved. In contrast to M88V and T45S, the D11N mutant virus particle was more stable than the wild-type virus, in spite of it also possessing a temperature sensitive growth phenotype. Overall, our data show how point substitutions in the capsid protein, which affect either the packing or the interaction at the protein-RNA interface, result in changes in virus stability.

Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment.

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.

A comparison of techniques for detecting Invertebrate iridescent virus 6.

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.

A possible iridovirus in erythrocytes of Bufo marinus in Costa Rica.

Icosahedral viral particles were found in the cytoplasm of erythrocytes and splenic reticular cells of a marine toad (Bufo marinus) collected from Costa Rica. Capsids had a maximum diameter of 312 nm and a spherical core with biphasic electron density. Viruses in erythrocytes were associated with cytoplasmic assembly areas and vacuoles in cytoplasm. Nuclei had finely granular material of decreased electron density located centrally, but contained no viral particles. A group of unenveloped viral particles was seen extracellularly in a splenic vessel. The virus was consistent with an iridovirus. In a blood smear stained with Giemsa round basophilic bodies with average diameters of 1.70 microns and morphologically similar to Pirhemocyton sp. were seen in the cytoplasm of erythrocytes and occasionally in the cytoplasm of monocytes or extracellularly. Erythrocytes containing these bodies had vacuoles and irregular pale-staining areas in the cytoplasm and pale-staining areas in the nucleus. These changes corresponded to the viral particles, assembly areas, vacuoles and nuclear changes at the ultrastructural level.

[Natural microbial control of crickets populations (Orthoptera: Gryllotalpidae: Scapteriscus borellii): regulation of populations aggregated in time and space]. / Controle microbiano natural de populações de paquinhas (Orthoptera: Gryllotalpidae: Scapteriscus borellii): regulação de populações agregadas no tempo e no espaço.

From 1983 through 1988, a total of 1,762 collections, containing 31,312 individuals of the mole cricket, Scapteriscus borellii, were made, principally in the State of São Paulo, Brazil. Collections were found to fit a negative binomial distribution both as whole and when divided into monthly collections. In these collections, an iridovirus, a entomogenous nematode, and the fungi Metarhizium anisopliae, Beauveria bassiana, Paecilomyces sp., and Entomophtora sp., were found to be agents of natural mortality, although usually as endozootics and relatively rarely as epizootics and panzootics. As a group, these diseases were also distributed in a binomial negative. These data suggest that the temporal and spatial aggregations of the mole crickets, produced by high rates of migration among suitable habitats, are adaptations to outbreaks of epidemics, which also serve as mole cricket population regulators. These ideas are develop and derived from simple mathematical models of population change.

Controle microbiano natural de populaçoës de paquinhas (Orthoptera: Gryllotalpidae: Scapteriscus borellii): regulaçäo de populaçoës agregadas no tempo e no espaço / Natural microbial control of crickets populations (Orthoptera: Gryllotalpidae: Scapteriscus borellii): regulations aggregated in time and space

Durante levantamentos realizados de 1983 a 1988, um total de 1.762 coletas, totalizando 3.312 indivíduos da paquinha, Scapteriscus borellii, foram feitas, principalmente no Estado de Säo Paulo, Brasil. As coletas ajustavam-se bem na totalidade, a uma distribuiçäo binomial negativa e também quando decomposta a coletas mensais. Das coletas, um iridovirus, um nematóide entomogênico, e os fungos Metarhizium anisopliae, Beauveria bassiana, Paecilomyces sp., Aspergillus sp., Sorsporella sp., e Entomophtora sp., foram encontrados como agentes de mortalidade natural, ainda que como endozoóticos e relativamente rara como epizoóticos ou panzoóticos. Como um conjunto, as doenças também foram distribuídas em forma binomial negativa. Estes dados implicam que as agregaçöes temporais e espaciais das paquinhas, determinadas por taxas elevadas de migraçäo entre habitats apropriados, säo respostas para enfrentar os surtos de doenças que também servem como reguladoras dos níveis populacionais da paquinha. Essas idéias säo desenvolvidas e derivadas de modelos matemáticos simples de mudanças de populaçöes