Absence of norovirus contamination in shellfish harvested and commercialized in the Northeast coast of Brazil.

Norovirus (NoV) is the main cause of gastroenteritis outbreaks worldwide. Although NoV spreads mainly from person to person, it is estimated that a large proportion of NoV outbreaks are caused by foodborne transmission. Bivalve mollusks are one of the most important foods involved in NoV transmission to humans. Little is known about NoV prevalence in shellfish harvested and commercialized in Brazil. The aim of this study was to map, for the first time, the distribution of NoV contamination in oysters and mussels harvested and commercialized in the coast of Pernambuco state, northeast Brazil. A total of 380 mollusks (260 oysters and 120 mussels) were collected between February and August 2017 either directly from harvesting areas or obtained from beach vendors at 17 sites in Pernambuco. Samples were processed and tested for NoV contamination using a SYBR Green real-time PCR assay. All samples were negative for NoV GI or GII contamination, suggesting a low risk of NoV contamination from this food source during the study period. Additional surveys in different areas of the Brazilian coast are warranted to monitor the risk of NoV infection upon seafood consumption.

Absence of norovirus contamination in shellfish harvested and commercialized in the Northeast coast of Brazil

Norovirus (NoV) is the main cause of gastroenteritis outbreaks worldwide. Although NoV spreads mainly from person to person, it is estimated that a large proportion of NoV outbreaks are caused by foodborne transmission. Bivalve mollusks are one of the most important foods involved in NoV transmission to humans. Little is known about NoV prevalence in shellfish harvested and commercialized in Brazil. The aim of this study was to map, for the first time, the distribution of NoV contamination in oysters and mussels harvested and commercialized in the coast of Pernambuco state, northeast Brazil. A total of 380 mollusks (260 oysters and 120 mussels) were collected between February and August 2017 either directly from harvesting areas or obtained from beach vendors at 17 sites in Pernambuco. Samples were processed and tested for NoV contamination using a SYBR Green real-time PCR assay. All samples were negative for NoV GI or GII contamination, suggesting a low risk of NoV contamination from this food source during the study period. Additional surveys in different areas of the Brazilian coast are warranted to monitor the risk of NoV infection upon seafood consumption.

Seroprevalence of selected flaviviruses in free-living and captive capuchin monkeys in the state of Pernambuco, Brazil.

Mosquito-borne diseases such as dengue, yellow fever and, more recently, Chikungunya virus (CHIKV) and Zika virus (ZIKV) have a great impact in the public health. In addition, the presence of such viruses might have an impact on wild animal conservation as well as their possible role as animal reservoir. Here, we performed a serological survey searching for antibodies against a panel of flaviviruses [ZIKV, Dengue virus (DENV), Yellow Fever virus (YFV), West Nile virus (WNV), Saint Louis Encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV)] using plaque reduction neutralization test (PRNT90 ) in both free-ranging and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus). Captive and free-living monkeys were sampled between June 2015 and January 2016 in the state of Pernambuco, including in the border with State of Paraíba, the epicentre of the ZIKV epidemics in Brazil. We have found neutralizing antibodies for ZIKV, DENV-1, DENV-2, DENV-3, DENV-4, YFV, ILHV and SLEV in both S. flavius and S. libidinosus samples. No positives samples were found for ROCV and WNV. Our results suggest that these flaviviruses might be circulating in capuchin monkey in the studied region. The possible presence of these viruses represents a risk for public health, as well as for animal conservation, especially for S. flavius which is a critically endangered species, facing high risk of extinction.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus.

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the N(pro) and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Immune transcript variations among Aedes aegypti populations with distinct susceptibility to dengue virus serotype 2.

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.

Microparticle-mediated RNA immunization against bovine viral diarrhea virus.

Infectious transcripts from the full-length infectious clone of the NADL strain of bovine viral diarrhea virus (BVDV) were used to vaccinate cattle and sheep against BVDV. In vitro synthesized RNA delivered by microparticle bombardment with a Helios Gene Gun initiated replication of BVDV and consequently induced humoral immunity against type I BVDV (serum neutralization titers, SNT > 2(12)) and type II BVDV (SNT > 2(7)). The quality and long-term stability of the RNA-carrier complexes was assessed by microparticle bombardment of tissue culture monolayers. The RNA cartridges were found to be stable for at least 8 months upon storage. This is the first report on successful RNA vaccination of large ruminants.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus.

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3. 1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes

Clinical, pathological and antigenic aspects of bovine viral diarrhea virus (BVDV) type 2 isolates identified in Brazil.

Nucleotide sequencing and phylogenetic analysis of Brazilian bovine viral diarrhea virus (BVDV) field isolates identified four viruses belonging to the genotype 2. Comparison of 5' UTR sequences from these isolates to those of North American BVDV type 2 revealed genomic variations that correlated with the geographic origins of the isolates. Two of the Brazilian type 2 viruses were isolated from clinical cases of gastroenteric/respiratory disease and two were isolated from healthy bovine fetuses. The clinical cases affected young animals (8- and 18-months-old) and were characterized by diarrhea, respiratory signs, extensive oral and digestive tract erosions, conjunctival and vulvar congestion, occasional digestive bleeding and vulvar and heart petechial hemorrhage. Antigenic analysis of these isolates with a panel of 10 monoclonal antibodies revealed marked antigenic differences in the major envelope glycoprotein, gp53/E2, compared to standard laboratory and vaccine BVDV strains. In addition, virus-specific antisera raised to Brazilian BVDV type 2 viruses displayed very low serological cross-reactivity with standard BVDV type 1 strains. Differences up to 64-fold in cross-neutralization titers were observed between BVDV type 1 and Brazilian BVDV type 2 isolates. The identification of BVDV type 2 among Brazilian cattle may have important implications for epidemiological studies, diagnostic and immunization strategies. Furthermore, the low neutralizing activity of BVDV type 1 antisera against the recently identified Brazilian BVDV type 2 isolates raises the question about the degree of protection conferred by BVDV vaccines, most of them based on a single type 1 strain.

Diversidade antigênica de amostras de vírus da diarréia viral bovina isoladas no Brasil: implicaçöes para o diagnóstico e estratégias de imunizaçäo / Antigenic diversity of Brazilian isolates of bovine viral diarrhea virus (BVDV): implications for diagnosis and immunization strategies

Seqüenciamento e análise filogenética de 17 amostras do vírus da diarréia viral bovina (BVDV) isoladas no Brasil identificaram quatro amostras (23,5 por cento) do genótipo 1a (BVDV-1a), nove amostras (52,9 por cento) do genótipo 1b (BVDV tipo 1b) e quatro amostras (23,5 por cento) do genótipo 2 (BVDV tipo 2). As amostras brasileiras de BVDV tipo 2 apresentaram-se genotipicamente distintas dos BVDV tipo 2 até entäo identificados na América do Norte e Europa, sugerindo pertencerem a um novo subgenótipo. A caracterizaçäo antigênica dessas amostras por neutralizaçäo cruzada revelou reatividade sorológica muito reduzida com cepas vacinais do BVDV. O anti-soro produzido contra três cepas vacinais do BVDV apresentou atividade neutralizante muito reduzida contra várias amostras brasileiras de BVDV tipo 1 e 2. Diferenças de até 128 vezes nos títulos de anticorpos neutralizantes foram observadas entre cepas vacinais e amostras brasileiras do BVDV. Nos testes de soroneutralizaçäo (SN) contra o vírus dos tipos 1 e 2, de 1134 amostras testadas, 280 (24,7 por cento) possuiam anticorpos neutralizantes anti-BVDV e dessas, 215 (76,8 por cento) apresentaram atividade neutralizante contra ambos os vírus, 37 (13,2 por cento) reagiram apenas contra o BVDV tipo 2 e 28 amostras (10 por cento) foram positivas apenas contra o BVDV tipo 1. Esses resultados demonstram que testes de SN utilizando vírus de apenas um genótipo podem resultar em número significativo de falsos-negativos e indica a necessidade da formulaçäo de vacinas com amostras locais de BVDV e/ou contendo vírus dos dois genótipos