RESUMO

After initiation of an infective cycle, spread of virus infection can occur in two fundamentally different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, and (ii) virions can remain associated with infected cells, promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence of cell-associated transmission has accumulated for many different viruses, the ability of members of the genus Pestivirus to use this mode of transmission has not been reported. In the present study, we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV) (the type member of the pestiviruses) infection. To demonstrate direct cell-to-cell transmission of BVDV, we developed a cell coculture system that allowed us to prove direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46, indicating that cell-to-cell transmission of the virus involves the engagement of coreceptors on the target cell.IMPORTANCE BVDV causes one of the most economically important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus, leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected if animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows the virus to overcome barriers to free virus dissemination, such as antibodies or epithelial barriers. Here we show that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid in the design of effective control strategies.

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RESUMO

Bovine viral diarrhea virus 1, reclassified as Pestivirus A, causes an economically important cattle disease that is distributed worldwide. Pestivirus A may cause persistent infection in that calves excrete the virus throughout their lives, spreading the infection in the herd. Many persistently infected (PI) calves die in the first 2 years of life from mucosal disease (MD) or secondary infections, probably as a consequence of virus-induced immune depression. Here, high-throughput sequencing (HTS) was applied for evaluation of the total virome in sera of (i) PI calves displaying clinically apparent MD (n = 8); (ii) PI calves with no signs of MD (n = 8); and (iii) control, Pestivirus A-free calves (n = 8). All the groups were collected at the same time and from the same herd. Serum samples from calves in each of the groups were pooled, submitted to viral RNA/DNA enrichment, and sequenced by HTS. Viral genomes of Pestivirus A, Ungulate erythroparvovirus 1, bosavirus (BosV), and hypothetical circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses were identified. Specific real-time PCR assays were developed to determine the frequency of occurrence of such viruses in each of the groups. The absolute number of distinct viral genomes detected in both PI calf groups was higher than in the control group, as revealed by higher number of reads, contigs, and genomes, representing a wider range of taxons. Genomes representing members of the family Parvoviridae, such as U. erythroparvovirus 1 and BosV, were most frequently detected in all the three groups of calves. Only in MD-affected PI calves, we found two previously unreported Hypothetical single-stranded DNA genomes clustered along with CRESS-DNA viruses. These findings reveal that parvoviruses were the most frequently detected viral genomes in cattle serum; its frequency of detection bears no statistical correlation with the status of calves in relation to Pestivirus A infection, since clinically normal or MD-affected/non-affected PI calves were infected with similar U. erythroparvovirus 1 genome loads. Moreover, MD-affected PI calves were shown to support viremia of CRESS-DNA viral genomes; however, the meaning of such correlation remains to be established.

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The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.

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Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.(AU)

Vacas prenhes infectadas com isolados não-citopáticos (NCP) do vírus da diarréia viral bovina (BVDV) entre os dias 40 e 120 de gestação frequentemente produzem bezerros imunotolerantes, persistentemente infectados (PI). Este artigo relata a caracterização de bezerros PI produzidos experimentalmente, pela inoculação de vacas prenhes com um pool de isolados brasileiros de BVDV-1 (n=2) e BVDV-2 (n=2) entre os dias 60 e 90 de gestação. Dois bezerros nasceram positivos para vírus, negativos para anticorpos, mas morreram aos dias 7 e 15 pós-nascimento, respectivamente. Outros seis bezerros nasceram saudáveis, soronegativos para o BVDV, replicaram e excretaram o vírus em secreções por até 210 dias. Análise antigênica dos vírus presentes nos bezerros PI com anticorpos monoclonais, ao nascimento e 30 dias após, revelou dois padrões de infecção. Três bezerros apresentavam apenas um dos vírus utilizados no desafio (BVDV-1 ou BVDV-2), enquanto outros três bezerros apresentavam co-infecção com mais de um vírus. Além disso, testando-se o sangue dos animais PI com um PCR capaz de diferenciar entre BVDV-1 e BVDV-2 observou-se a presença concomitante de vírus dos dois genótipos (BVDV-1 e BVDV-2) nestes três animais. Esses resultados indicam que infecção persistente - uma consequência bem conhecida da infecção fetal pelo BVDV - pode ser estabelecida concomitantemente por mais de um vírus, após infecção experimental. Nesse sentido, infecções persistentes mistas com isolados antigenicamente distintos podem ser úteis para o estudo das bases imunológicas e moleculares da imunotolerância e infecção persistente pelo BVDV.(AU)

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A pesquisa de animais persistentemente infectados (PI) pelo vírus da diarréia viral bovina (BVDV) foi realizada em 26 rebanhos bovinos, não vacinados contra o BVDV, localizados nos Estados de Minas Gerais e São Paulo, Brasil. Utilizando uma estratégia de amostragem, de cada rebanho foram obtidas cinco amostras de sangue de bezerros, entre 6 e 12 meses de idade, e os soros sanguíneos foram submetidos ao teste de virusneutralização (VN) para o BVDV-1 e o BVDV-2. Os rebanhos que apresentaram pelo menos três das cinco amostras reagentes a um dos genótipos do BVDV, e com títulos de anticorpos superiores a 128, foram selecionados para a pesquisa de animais PI. Em três rebanhos que apresentaram tal condição, foram colhidas amostras pareadas de sangue de todos os bovinos do rebanho, com intervalo de 30 dias entre as colheitas, e o soro sanguíneo foi submetido ao teste de VN para o BVDV-1 e o BVDV-2. Nas amostras não reagentes a pelo menos um dos genótipos do BVDV e naquelas provenientes de bovinos com menos de seis meses de idade, realizou-se a pesquisa do BVDV pela reação em cadeia da polimerase precedida pela transcrição reversa (RT-PCR). Dos rebanhos analisados, foram detectados dois animais PI a partir de amostras obtidas nas colheitas pareadas provenientes de um rebanho localizado no Estado de Minas Gerais.(AU)

The research on persistently infected (PI) animals with bovine viral diarrhoea virus (BVDV) was conducted in 26 cattle herds, which were not BVDV vaccinated, located in the states of Minas Gerais and São Paulo, Brazil. Using a sampling strategy, five samples of blood were collected from 6 to 12-month-old calves of each herd, and the blood sera were tested by virusneutralization test (VN) to BVDV-1 and BVDV-2. The herds that had at least three out of five samples reacting to one of the genotypes of BVDV and antibody titers greater than 128 were selected to PI animals research. In three of the herds that matched the before-mentioned criteria, paired blood samples were collected from all its individuals considering a collection interval of 30 days. The blood sera of these samples were VN tested against BVDV-1 and BVDV-2. In samples not reacting to at least one of the BVDV genotypes and also in those collected from calves of less than six months of age, virus research was undertaken by reverse transcription polymerase chain reaction (RT-PCR). From the examined herds, two PI animals were detected in paired samples obtained from a herd located in the state of Minas Gerais.(AU)

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RESUMO

A pesquisa de animais persistentemente infectados (PI) pelo vírus da diarréia viral bovina (BVDV) foi realizada em 26 rebanhos bovinos, não vacinados contra o BVDV, localizados nos Estados de Minas Gerais e São Paulo, Brasil. Utilizando uma estratégia de amostragem, de cada rebanho foram obtidas cinco amostras de sangue de bezerros, entre 6 e 12 meses de idade, e os soros sanguíneos foram submetidos ao teste de virusneutralização (VN) para o BVDV-1 e o BVDV-2. Os rebanhos que apresentaram pelo menos três das cinco amostras reagentes a um dos genótipos do BVDV, e com títulos de anticorpos superiores a 128, foram selecionados para a pesquisa de animais PI. Em três rebanhos que apresentaram tal condição, foram colhidas amostras pareadas de sangue de todos os bovinos do rebanho, com intervalo de 30 dias entre as colheitas, e o soro sanguíneo foi submetido ao teste de VN para o BVDV-1 e o BVDV-2. Nas amostras não reagentes a pelo menos um dos genótipos do BVDV e naquelas provenientes de bovinos com menos de seis meses de idade, realizou-se a pesquisa do BVDV pela reação em cadeia da polimerase precedida pela transcrição reversa (RT-PCR). Dos rebanhos analisados, foram detectados dois animais PI a partir de amostras obtidas nas colheitas pareadas provenientes de um rebanho localizado no Estado de Minas Gerais.

The research on persistently infected (PI) animals with bovine viral diarrhoea virus (BVDV) was conducted in 26 cattle herds, which were not BVDV vaccinated, located in the states of Minas Gerais and São Paulo, Brazil. Using a sampling strategy, five samples of blood were collected from 6 to 12-month-old calves of each herd, and the blood sera were tested by virusneutralization test (VN) to BVDV-1 and BVDV-2. The herds that had at least three out of five samples reacting to one of the genotypes of BVDV and antibody titers greater than 128 were selected to PI animals research. In three of the herds that matched the before-mentioned criteria, paired blood samples were collected from all its individuals considering a collection interval of 30 days. The blood sera of these samples were VN tested against BVDV-1 and BVDV-2. In samples not reacting to at least one of the BVDV genotypes and also in those collected from calves of less than six months of age, virus research was undertaken by reverse transcription polymerase chain reaction (RT-PCR). From the examined herds, two PI animals were detected in paired samples obtained from a herd located in the state of Minas Gerais.

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RESUMO

Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural.(AU)

Assuntos

RESUMO

Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural.

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