Otimização da soroneutralização com diferentes tipos e subtipos de herpesvírus bovino e sua aplicação à epidemiologia

No presente estudo, buscou-se avaliar quais seriam as cepas de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) seriam mais adequadas para uso como vírus de confrontação em testes de soroneurtralização (SN). Oitocentas e dez amostras de soros de duas regiões geograficamente distintas foram avaliadas à SN frente a seis diferentes cepas virais, incluindo tipos e subtipos diversos (BoHV-1.1: EVI123/98 e Los Angeles; BoHV-1.2a: SV265/96; BoHV-5a: EVI88/95; BoHV-5b: A663 e BoHV-5c: ISO95/97). A maior sensibilidade foi revelada pelo somatório de soropositivos identificados com as seis cepas utilizadas no estudo. Uma combinação de quatro vírus (BoHV-1.1: LA e EVI123/98, BoHV-5a: EVI88/95 e BoHV-5b: A663) foi capaz de detectar 99,1% das amostras soropositivas. Estes quatro vírus foram selecionados para serem utilizados em um levantamento soroepidemiológico, com o intuito de estimar a prevalência das infecções causadas pelos BoHV-1 e BoHV-5 no Estado do Rio Grande do Sul. Para tanto, soros de 2200 fêmeas bovinas adultas (>24 meses), representativos da população bovina do Rio Grande do Sul, foram submetidos à SN frente às quatro cepas virais escolhidas. Com essa combinação, a soroprevalência média das infecções por BoHV-1 e BoHV-5 encontrada foi de 29,2%. Além disso, analisando outros fatores que pudessem influenciar a prevalência das infecções, foram considerados fatores de risco o tipo de exploração corte, a ausência da prática de ordenha, o contato dos bovinos com ovinos/caprinos e animais silvestres, a venda de animais de reprodução, o uso de piquetes de parto/pós-parto, o uso de assistência veterinária privada e a criação de animais de raças europeias de corte. Os resultados obtidos no presente trabalho demonstraram que, para que a SN seja capaz de detectar animais soropositivos ao BoHV-1 e BoHV-5 com a máxima sensibilidade, o teste deve ser realizado com várias amostras de BoHV-1 e BoHV-5, não necessariamente de tipos ou subtipos diferentes, pois amostras do mesmo tipo de vírus apresentarrm diferentes sensibilidades. Além disso, as amostras de confrontação podem variar de acordo com a região geográfica de origem dos soros. Os resultados obtidos nesse levantamento revelam que anticorpos anti-BoHV-1 e anti-BoHV-5 encontram-se amplamente distribuidos nos rebanhos gaúchos. No entanto, não foi possível determinar a prevalência tipo-específica destas infecções com os testes realizados. Assim, a proporção de animais que estão infectados com o BoHV-1 ou com o BoHV-5 (ou ambos) na região examinada permanece desconhecida

Blood or serum collected on filter paper for detection of antibodies to bovine herpesvirus type 1 (BoHV-1)

Background: The method of collection as well as the packaging conditions in which samples are submitted to laboratories play a critical role on the acquisition of reliable results on diagnostic tests. Alternative methods however have been proposed, as the adsorption of blood or serum in filter paper. In this work, it was evaluated the viability of using serum or whole blood samples from bovines collected in filter paper for serological testing against bovine herpesvirus type 1 (BoHV-1). Materials, Methods & Results: One hundred and seven whole blood and serum samples were collected by standard methods. Serum neutralization test was used as a golden standard method for evaluation of the serum samples. The same samples of both whole blood and sera were also adsorbed on filter paper strips for further comparisons. Optimal conditions for serum and blood elution from filter paper were determined. Adsorbed samples on filter paper disks were eluted in PBS and subsequently diluted further with PBS 5% skimmed milk. The eluates were tested for antibodies to BoHV-1 in an indirect ELISA (iELISA) and matched with the results obtained by serum neutralization of standard serum samples. Comparison between results obtained by serum neutralization of standard serum samples and the ones from the iELISA of serum eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 95, 94, 80, 99%, respectively, and a correlation coefficient (κ) of 0.83. Comparison between the results of serum neutralization of standard serum samples and the ones from the iELISA of blood eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 91, 97, 87, 98% , respectively, and a correlation coefficient (κ) of 0.86. Standard serum samples were also tested in the iELISA and the results compared with those of iELISA from samples eluted from filter paper. Comparison of the results from iELISA between serum samples and serum eluted from filter paper resulted in sensitivity, specificity, positive and negative predictive values of 91, 81, 69, 95%, respectively, and a correlation coefficient (κ) of 0.66. The comparison of iELISA results between standard serum and blood samples eluted from filter paper resulted, in sensitivity, specificity, positive and negative predictive values of 78, 92, 82, 89%, respectively, and a correlation coefficient (κ) of 0.70. Fifty samples collected on filter paper were retested eight months later in order to determine whether those would retain its viability; both sensitivity and specificity remained unaltered. Discussion: Sampling on filter paper has been successfully described for antibody detection in a number of diseases such as Aujeszky’s disease virus and Newcastle disease virus. In this work, it has been demonstrated that both blood and sera collected in filter paper can be used for submission of samples aiming detection of antibodies to BoHV-1 in an iELISA, without significant loss of sensitivity and specificity. Submission of samples on filter paper is a practical and economical alternative as no special conditions are required for storaging and transporting. This method also enables the collection of samples from distant places assuring its quality for serological test

Caracterização genética de isolados brasileiros de circovírus suíno tipo 2

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Vacinação com herpesvírus bovino tipo 1 (BHV-1) não protege coelhos contra encefalites por BHV-5

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Torque Teno Sus Virus (TTSuV) in Cell Cultures and Trypsin

Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells

Serum neutralization optimization with different bovine herpesviruses types and subtypes and its epidemiology application

No presente estudo, buscou-se avaliar quais seriam as cepas de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) seriam mais adequadas para uso como vírus de confrontação em testes de soroneurtralização (SN). Oitocentas e dez amostras de soros de duas regiões geograficamente distintas foram avaliadas à SN frente a seis diferentes cepas virais, incluindo tipos e subtipos diversos (BoHV-1.1: EVI123/98 e Los Angeles; BoHV-1.2a: SV265/96; BoHV-5a: EVI88/95; BoHV-5b: A663 e BoHV-5c: ISO95/97). A maior sensibilidade foi revelada pelo somatório de soropositivos identificados com as seis cepas utilizadas no estudo. Uma combinação de quatro vírus (BoHV-1.1: LA e EVI123/98, BoHV-5a: EVI88/95 e BoHV-5b: A663) foi capaz de detectar 99,1% das amostras soropositivas. Estes quatro vírus foram selecionados para serem utilizados em um levantamento soroepidemiológico com o intuito de estimar a prevalência das infecções causadas pelos BoHV-1 e BoHV-5 no Estado do Rio Grande do Sul. Para tanto, soros de 2200 fêmeas bovinas adultas (>24 meses), representativos da população bovina do Rio Grande do Sul, foram submetidos à SN frente às quatro cepas virais escolhidas. om esta combinação, a soroprevalência média das infecções por BoHV-1 e BoHV-5 encontrada foi de 29,2%. Além disso, analisando outros fatores que pudessem influenciar a prevalência das infecções, foram considerados fatores de risco o tipo de exploração corte, a ausência da prática de ordenha, o contato dos bovinos com ovinos/caprinos e animais silvestres, a venda de animais de reprodução, o uso de piquetes de parto/pós-parto, o uso de assistência veterinária privada e a criação de animais de raças européias de corte. Os resultados obtidos no presente trabalho demonstraram que, para que a SN seja capaz de detectar animais soropositivos ao BoHV-1 e BoHV-5 com a máxima sensibilidade, o teste deve ser realizado com várias amostras de BoHV-1 e BoHV-5, não necessariamente de tipos ou subtipos diferentes, pois amostras do mesmo tipo de vírus apresentarrm diferentes sensibilidades. Além disso, as amostras de confrontação podem variar de acordo com a região geográfica de origem dos soros. Os resultados obtidos nesse levantamento revelam que anticorpos anti-BoHV-1 e anti-BoHV-5 encontram-se amplamente distribuidos nos rebanhos gaúchos. No entanto, não foi possível determinar a prevalência tipo-específica destas infecções com os testes realizados. Assim, a proporção de animais que estão infectados com o BoHV-1 ou com o BoHV-5 (ou ambos) na região examinada permanece desconhecida.In this study a search was carried out to determine which of a number of available strains/isolates of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) would be more suitable for use as challenge virus in serum neutralization (SN) tests. Eight hundred and ten bovine serum samples collected from two geographically distinct regions were evaluated in SN tests against six BoHVs of different types and subtypes (BoHV-1.1: EVI123/98 and Los Angeles; BoHV-1.2a: SV265/96; BoHV-5a: EVI88/95; BoHV-5b: A663 and BoHV-5c: ISO95/97). The highest sensitivity was achieved when the SN-positive sera obtained with the six different viruses were added. A combination of four viruses (BoHV-1.1 LA, and EVI123/98, BoHV-5a EVI88/95 and BoHV-5b A663), was able to detect 99.1% of the seropositive samples. These four viruses were selected to carry out a seroepidemiological survey to estimate BoHV-1 and BoHV-5 prevalence in the state of Rio Grande do Sul. In order to achieve that, sera from 2,200 bovine female cows (>24 months-old), representative of the bovine population of Rio Grande do Sul state were tested on SN tests against the four selected viruses. With such combination, seroprevalence of BoHV-1 and BoHV-5 infections was 29.2%. After examining potential factors that might affect the prevalence of such infections, the following were considered as significant risk factors: the type of exploitation (beef cattle > dairy), use of milking procedures, concomitant presence of sheep, goats or wild animals in farm; sale of animals for reproductive purposes; use of pre and postparturition paddocks; use of private veterinary assistance and farming of European beef breeds. The results obtained here indicate that for the SN test to provide the highest sensitivity in detecting BoHV-1 and BoHV-5 seroposivive animals, it must be performed against a number of BoHV-1 and BoHV-5 strains, though not necessarily of different types and subtypes as viruses within a same subtype may display different sensitivities. Besides, challenge viruses may vary for geographically distinct areas. The serological survey performed with four distinct bovine herpesviruses reveal that antibodies to BoHV-1 and BoHV-5 are widely distributed among cattle flocks in Rio Grande do Sul. However, it was not possible to determine type-specific prevalence with the tests performed. Thus, the proportion of cattle actually infected with either BoHV-1 or BoHV-5 (or both) in the examined region remains undetermined

Serum neutralization optimization with different bovine herpesviruses types and subtypes and its epidemiology application

No presente estudo, buscou-se avaliar quais seriam as cepas de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) seriam mais adequadas para uso como vírus de confrontação em testes de soroneurtralização (SN). Oitocentas e dez amostras de soros de duas regiões geograficamente distintas foram avaliadas à SN frente a seis diferentes cepas virais, incluindo tipos e subtipos diversos (BoHV-1.1: EVI123/98 e Los Angeles; BoHV-1.2a: SV265/96; BoHV-5a: EVI88/95; BoHV-5b: A663 e BoHV-5c: ISO95/97). A maior sensibilidade foi revelada pelo somatório de soropositivos identificados com as seis cepas utilizadas no estudo. Uma combinação de quatro vírus (BoHV-1.1: LA e EVI123/98, BoHV-5a: EVI88/95 e BoHV-5b: A663) foi capaz de detectar 99,1% das amostras soropositivas. Estes quatro vírus foram selecionados para serem utilizados em um levantamento soroepidemiológico com o intuito de estimar a prevalência das infecções causadas pelos BoHV-1 e BoHV-5 no Estado do Rio Grande do Sul. Para tanto, soros de 2200 fêmeas bovinas adultas (>24 meses), representativos da população bovina do Rio Grande do Sul, foram submetidos à SN frente às quatro cepas virais escolhidas. om esta combinação, a soroprevalência média das infecções por BoHV-1 e BoHV-5 encontrada foi de 29,2%. Além disso, analisando outros fatores que pudessem influenciar a prevalência das infecções, foram considerados fatores de risco o tipo de exploração corte, a ausência da prática de ordenha, o contato dos bovinos com ovinos/caprinos e animais silvestres, a venda de animais de reprodução, o uso de piquetes de parto/pós-parto, o uso de assistência veterinária privada e a criação de animais de raças européias de corte. Os resultados obtidos no presente trabalho demonstraram que, para que a SN seja capaz de detectar animais soropositivos ao BoHV-1 e BoHV-5 com a máxima sensibilidade, o teste deve ser realizado com várias amostras de BoHV-1 e BoHV-5, não necessariamente de tipos ou subtipos diferentes, pois amostras do mesmo tipo de vírus apresentarrm diferentes sensibilidades. Além disso, as amostras de confrontação podem variar de acordo com a região geográfica de origem dos soros. Os resultados obtidos nesse levantamento revelam que anticorpos anti-BoHV-1 e anti-BoHV-5 encontram-se amplamente distribuidos nos rebanhos gaúchos. No entanto, não foi possível determinar a prevalência tipo-específica destas infecções com os testes realizados. Assim, a proporção de animais que estão infectados com o BoHV-1 ou com o BoHV-5 (ou ambos) na região examinada permanece desconhecida.In this study a search was carried out to determine which of a number of available strains/isolates of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) would be more suitable for use as challenge virus in serum neutralization (SN) tests. Eight hundred and ten bovine serum samples collected from two geographically distinct regions were evaluated in SN tests against six BoHVs of different types and subtypes (BoHV-1.1: EVI123/98 and Los Angeles; BoHV-1.2a: SV265/96; BoHV-5a: EVI88/95; BoHV-5b: A663 and BoHV-5c: ISO95/97). The highest sensitivity was achieved when the SN-positive sera obtained with the six different viruses were added. A combination of four viruses (BoHV-1.1 LA, and EVI123/98, BoHV-5a EVI88/95 and BoHV-5b A663), was able to detect 99.1% of the seropositive samples. These four viruses were selected to carry out a seroepidemiological survey to estimate BoHV-1 and BoHV-5 prevalence in the state of Rio Grande do Sul. In order to achieve that, sera from 2,200 bovine female cows (>24 months-old), representative of the bovine population of Rio Grande do Sul state were tested on SN tests against the four selected viruses. With such combination, seroprevalence of BoHV-1 and BoHV-5 infections was 29.2%. After examining potential factors that might affect the prevalence of such infections, the following were considered as significant risk factors: the type of exploitation (beef cattle > dairy), use of milking procedures, concomitant presence of sheep, goats or wild animals in farm; sale of animals for reproductive purposes; use of pre and postparturition paddocks; use of private veterinary assistance and farming of European beef breeds. The results obtained here indicate that for the SN test to provide the highest sensitivity in detecting BoHV-1 and BoHV-5 seroposivive animals, it must be performed against a number of BoHV-1 and BoHV-5 strains, though not necessarily of different types and subtypes as viruses within a same subtype may display different sensitivities. Besides, challenge viruses may vary for geographically distinct areas. The serological survey performed with four distinct bovine herpesviruses reveal that antibodies to BoHV-1 and BoHV-5 are widely distributed among cattle flocks in Rio Grande do Sul. However, it was not possible to determine type-specific prevalence with the tests performed. Thus, the proportion of cattle actually infected with either BoHV-1 or BoHV-5 (or both) in the examined region remains undetermined

Dynamics of the in vitro emergence of escape mutants of the peste des petits ruminants virus (PPRV) to interfering RNAs targeting the nucleoprotein gene : implications for therapeutics

Les membres du genre Morbillivirus, famille Paramyxoviridae sont responsables de graves maladies chez l'homme et les animaux, comme la rougeole, la peste bovine (RP) et la peste des petits ruminants (PPR). Malgré l'existence de vaccins efficaces contre ces maladies, des traitements spécifiques sont souhaitables. L'inhibition de la réplication de ces virus peut-être acquise par interférence ARN (ARNi), un mécanisme d'inhibition post-transcriptionnel déclenché par des séquences courtes d'ARN double-brin (siARN). Le CIRAD a précédemment identifié 3 siARNs ciblant des régions conservées du gène de la nucléoprotéine virale capables d'inhiber au moins 80% de la réplication in vitro des virus de la rougeole, de la RP et de la PPR. Cependant, un problème majeur dans la stratégie d'ARNi est le risque d'apparition de virus résistants. Dans cette étude, nous avons évalué le risque d'apparition de mutants d'échappement du virus de la PPR sous pression de sélection de 3 siARNs appliqués seul ou en association après plusieurs transfections successives in vitro. Excepté pour la combinaison des 3 siARNs, le virus a échappé à l'ARNi après 3 à 20 passages consécutifs, avec des mutations simples ou multiples (synonymes ou pas) ou une délétion de 6 nucléotides dans la zone cible des siARN. Ces résultats mettent en évidence une plasticité génomique inattendue des morbillivirus surtout illustrée par cette délétion non-délétère d'une partie significative d'un gène viral essentiel, qui devrait être considérée comme un obstacle à l'utilisation de l'ARNi comme thérapie antivirale. Cependant, l'utilisation combinée de 3 siARNs peut être proposée pour diminuer le risque d'échappement aux siARNs.Viruses in the genus Morbillivirus, within the family Paramyxoviridae are responsible for severe humans and animal diseases, including measles, rinderpest (RP) and peste des petits ruminants (PPR). In spite of the existence of efficient vaccines against these diseases, specific treatments to be applied when the infection is already present are desirable. Inhibition of morbillivirus replication can be achieved by RNA interference (RNAi), a mechanism of post-transcriptional gene silencing triggered by small double-stranded RNA (siRNA). The CIRAD previously identified three siRNAs that target conserved regions of the essential gene encoding the viral nucleoprotein and are able to prevent in vitro at least 80% of the replication of measles, RP and PPR viruses . However, a major problem in RNAi is the important risk of emergence of escape mutants. In this study, we investigated the ability of PPR virus to escape the inhibition conferred by single or multiple siRNAs after several consecutive transfections in vitro. Except with the combination of the three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The mutations were characterized by either single or multiple punctual nucleotide mutations (synonymous or not) or a deletion of a stretch of 6 nucleotides into the siRNA target. These results demonstrate that the genomic plasticity of morbilliviruses, illustrated maily by this significant and no-deleterious deletion in an essential viral gene, should be considered as an obstacle to the use of RNAi in antiviral therapy. However, the combined use of three siRNAs can be proposed to prevent treatment failure with siRNAs

Dynamique de l'émergence in vitro des mutants d'échappement du virus de la peste des petits ruminants (PPRV) face à l'activité ARN interférente ciblant le gène de la nucléoprotéine (implications pour les stratégies thérapeutiques)

Les membres du genre Morbillivirus, famille Paramyxoviridae sont responsables de graves maladies chez l'homme et les animaux, comme la rougeole, la peste bovine (RP) et la peste des petits ruminants (PPR). Malgré l'existence de vaccins efficaces contre ces maladies, des traitements spécifiques sont souhaitables. L'inhibition de la réplication de ces virus peut-être acquise par interférence ARN (ARNi), un mécanisme d'inhibition post-transcriptionnel déclenché par des séquences courtes d'ARN double-brin (siARN). Le CIRAD a précédemment identifié 3 siARNs ciblant des régions conservées du gène de la nucléoprotéine virale capables d'inhiber au moins 80% de la réplication in vitro des virus de la rougeole, de la RP et de la PPR. Cependant, un problème majeur dans la stratégie d'ARNi est le risque d'apparition de virus résistants. Dans cette étude, nous avons évalué le risque d'apparition de mutants d'échappement du virus de la PPR sous pression de sélection de 3 siARNs appliqués seul ou en association après plusieurs transfections successives in vitro. Excepté pour la combinaison des 3 siARNs, le virus a échappé à l'ARNi après 3 à 20 passages consécutifs, avec des mutations simples ou multiples (synonymes ou pas) ou une délétion de 6 nucléotides dans la zone cible des siARN. Ces résultats mettent en évidence une plasticité génomique inattendue des morbillivirus surtout illustrée par cette délétion non-délétère d'une partie significative d'un gène viral essentiel, qui devrait être considérée comme un obstacle à l'utilisation de l'ARNi comme thérapie antivirale. Cependant, l'utilisation combinée de 3 siARNs peut être proposée pour diminuer le risque d'échappement aux siARNs.Viruses in the genus Morbillivirus, within the family Paramyxoviridae are responsible for severe humans and animal diseases, including measles, rinderpest (RP) and peste des petits ruminants (PPR). In spite of the existence of efficient vaccines against these diseases, specific treatments to be applied when the infection is already present are desirable. Inhibition of morbillivirus replication can be achieved by RNA interference (RNAi), a mechanism of post-transcriptional gene silencing triggered by small double-stranded RNA (siRNA). The CIRAD previously identified three siRNAs that target conserved regions of the essential gene encoding the viral nucleoprotein and are able to prevent in vitro at least 80% of the replication of measles, RP and PPR viruses . However, a major problem in RNAi is the important risk of emergence of escape mutants. In this study, we investigated the ability of PPR virus to escape the inhibition conferred by single or multiple siRNAs after several consecutive transfections in vitro. Except with the combination of the three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The mutations were characterized by either single or multiple punctual nucleotide mutations (synonymous or not) or a deletion of a stretch of 6 nucleotides into the siRNA target. These results demonstrate that the genomic plasticity of morbilliviruses, illustrated maily by this significant and no-deleterious deletion in an essential viral gene, should be considered as an obstacle to the use of RNAi in antiviral therapy. However, the combined use of three siRNAs can be proposed to prevent treatment failure with siRNAs.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF