The constitutive expression of the V gene of Parainfluenza virus 5 affects the growth properties of bovine herpesvirus 5

This study aimed to analyze the effect of the expression of Parainfluenza virus 5 (PIV5) V protein in bovine cells on the replication of Bovine herpesvirus 5 (BoHV-5). Growth properties of BoHV-5 were evaluated in parental and PIV5 transfected cells. In one-step growth experiments, the BoHV-5 reached higher titers at earlier time points in the transfected cells when compared to the parental cells. The mean plaque size produced by the BoHV-5 in transfected cells was larger than the parental cells. This indicated that the expression of the PIV5 V gene facilitated the release and cell-to-cell spread of BoHV-5 in bovine cells.

Bovine herpesviruses do not play a major role in the differential diagnosis of rabies in cattle in southern Brazil

Background: Rabies has long been recognized as the major cause of encephalitis in cattle in Latin American countries. It has been estimated that nearly 50.000 cattle heads per year are lost due to encephalitis in that subcontinent, with a significant economic impact on cattle productive chains. In Brazil only, 2.500 to 3.000 cattle heads are estimated to be lost every year due to rabies. However, it is believed that rabies incidence in cattle is much larger, since usually only a few samples from affected animals in disease outbreaks are submitted to diagnostic laboratories. Rabies encephalitis is promptly and accurately diagnosed; however, particularly when rabies is excluded as causa mortis, the agent responsible for neurological disease of infectious origin often remains undetermined. Two bovine herpesviruses (BoHVs), bovine herpesvirus type 1 (BoHV-1) and bovine herpesvirus type 5 (BoHV-5) are major pathogens of cattle which are widely disseminated in Brazil. As usual in herpesvirus' biology, these tend to infect a large number of hosts and establish lifelong latent infections which may occasionally be reactivated. Both viruses, particularly BoHV-5, are often recovered from cases of neurological disease in cattle. The participation of BoHVs in the differential diagnosis of rabies must be evaluated. Besides, there might be associations between the occurrence of rabies and BoHV infections that deserve investigation. The aim of this study was to investigate whether bovine herpesvirus 1 and 5 would play a significant role in cases of neurological disease where rabies was the presumptive clinical diagnosis. In addition, associations between the occurrence of rabies and BoHV infections were searched for. The approach adopted for conducting such investigations was based on the search for viral nucleic acids as well as classical virus isolation on tissues of cattle submitted to rabies diagnosis over a two-year period, including rabies-positive and rabies-negative specimens. Materials, Methods & Results: Brain tissue samples of 101 cattle originally submitted to rabies diagnosis were collected over a two year period (2009-2010) from various municipalities within the state of Rio Grande do Sul, Brazil. Thirty nine of these samples had the diagnosis of rabies confirmed by standard laboratory diagnostic methods. Aliquots of tissues were submitted to DNA extraction and examined in search for genomes of bovine herpesviruses (BoHV) types 1 (BoHV-1) and 5 (BoHV-5) by as well as for infectious virus. Bovine herpesvirus genomes were detected in 78/101 (77.2%) samples, in which BoHV-1 genomes were detected in 26/78 (25.7%), BoHV-5 genomes in 22/78 (21.8%) and mixed BoHV infections (BoHV-1 and BoHV-5 genomes) were detected in 30/101 (29.7%) samples. In the 39 samples with confirmed rabies diagnosis, BoHV-1 DNA was detected in 9/39 (23%), BoHV-5 DNA in 6/39 (15.4%) and mixed infections with both BoHV types in 16/39 (41%) samples. However, no infectious herpesvirus was recovered from any of the specimens examined. Discussion: The high prevalence of BoHV1 and BoHV-5 infections was evidenced in the sampled population, but the absence of infectious BoHVs indicate that these were not associated to the occurrence of the cases of encephalitis where rabies was the primary suspicion. In addition, no association was detected between occurrence of rabies and detection of BoHVs, since the frequency of detection of herpesvirus genomes did not significantly differ between rabies-positive and rabies-negative samples. The detection of BoHV DNA in scattered areas of the brain with no infectious virus suggests that latency may take place in different regions of the brain.

Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: applications on detection, sequencing and virus isolation.

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93-99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10(5.55) TCID(50)/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.

Transfecção de DNA de BoHV-5 com fosfato de cálcio em diferentes tipos de cultivos celulares / Transfection of BoHV-5 DNA with calcium phosphate in different types of cell culture

As condições para uma transfecção eficaz pelo método de fosfato de cálcio podem variar substancialmente. Neste estudo foi testado o tipo de cultivo celular mais apropriado para transfecção de DNA de herpesvírus bovino tipo 5 (BoHV-5) por esta técnica. Primeiramente foi avaliada a capacidade de multiplicação do BoHV-5 em três diferentes tipos de células. Para isto, a concentração viral foi calculada pelo método de dose infectante para cultivo celular / 50% (TCID50/mL). O BoHV-5 foi titulado em células de linhagem de rim de macaco verde Africano (VERO), células de linhagem de rim de suíno (PKsC3), células primárias de testículos de terneiro (TT) e células de linhagem de rim de bovino (MDBK). Em células VERO e PKsC3 a concentração viral máxima obtida foi 103,3 TCID50/mL, enquanto que em células MDBK o título foi de 106,8 TCID50/mL, similar ao encontrado para as células TT. Para avaliar a eficácia da transfecção nessas células, foi utilizado DNA plasmideal expressando um gene repórter (EGFP). Os resultados mostraram um valor de 4,5% de células fluorescentes para células TT, de 2,9% para PKsC3, de 0,5% para VERO e de 0,05% para MDBK. Quando células PKsC3 e TT foram transfectadas com 0,5 μg de DNA purificado de BoHV-5, somente as células TT apresentaram resultado positivo, com duas placas virais por poço. Para investigar a influência da concentração de DNA viral, células TT foram transfectadas com 0,5 μg e 1 μg de DNA purificado de BoHV-5, produzindo duas e quatro placas, respectivamente. Nas condições experimentais testadas neste estudo os resultados indicam que as células TT poderiam ser boas candidatas para serem utilizadas para a transfecção de DNA de BoHV-5.(AU)

The conditions for an efficacious transfection by calcium phosphate method can vary substantially. In this study the most appropriate cell type for transfection of bovine herpesvirus type 5 (BoHV-5) DNA using this technique was tested. It was first evaluated the capacity of BoHV-5 multiplication in three different cell types. For this purpose virus concentration was calculated by tissue culture infectious dose 50% assay (TCID50/mL). BoHV-5 was titrated in kidney cells from African green monkey (VERO), cell clone of pig kidney (PKsC3), primary cell culture of calf testis (TT) and bovine kidney cells (MDBK). In Vero and PK15 cells maximum virus concentration reached 103,3 TCID50/mL, whereas in MDBK cells the titre was 106,8 TCID50/mL, similar to that of TT cells. In order to evaluate the transfection efficacy in these cells, plasmid DNA expressing a reporter gene (EGFP) was used. Results showed 4.5% fluorescent cells for TT, 2.9% for PKsC3, 0.5% for VERO and 0.05% for MDBK cells. When PKsC3 and TT cells were transfected with 0.5 mg of BoHV-5 purified DNA, only TT cells gave positive result with two plaques per well. To investigate the influence of viral DNA concentration, TT cells were then transfected with 0.5 mg and 1.0 mg of purified BoHV-5 DNA producing two and four plaques, respectively. Under experimental conditions tested in this study the results indicate that TT cells could be good candidates to be used for transfection of BoHV-5 DNA.(AU)

Transfecção de DNA de BoHV-5 com fosfato de cálcio em diferentes tipos de cultivos celulares / Transfection of BoHV-5 DNA with calcium phosphate in different types of cell culture

As condições para uma transfecção eficaz pelo método de fosfato de cálcio podem variar substancialmente. Neste estudo foi testado o tipo de cultivo celular mais apropriado para transfecção de DNA de herpesvírus bovino tipo 5 (BoHV-5) por esta técnica. Primeiramente foi avaliada a capacidade de multiplicação do BoHV-5 em três diferentes tipos de células. Para isto, a concentração viral foi calculada pelo método de dose infectante para cultivo celular / 50% (TCID50/mL). O BoHV-5 foi titulado em células de linhagem de rim de macaco verde Africano (VERO), células de linhagem de rim de suíno (PKsC3), células primárias de testículos de terneiro (TT) e células de linhagem de rim de bovino (MDBK). Em células VERO e PKsC3 a concentração viral máxima obtida foi 103,3 TCID50/mL, enquanto que em células MDBK o título foi de 106,8 TCID50/mL, similar ao encontrado para as células TT. Para avaliar a eficácia da transfecção nessas células, foi utilizado DNA plasmideal expressando um gene repórter (EGFP). Os resultados mostraram um valor de 4,5% de células fluorescentes para células TT, de 2,9% para PKsC3, de 0,5% para VERO e de 0,05% para MDBK. Quando células PKsC3 e TT foram transfectadas com 0,5 μg de DNA purificado de BoHV-5, somente as células TT apresentaram resultado positivo, com duas placas virais por poço. Para investigar a influência da concentração de DNA viral, células TT foram transfectadas com 0,5 μg e 1 μg de DNA purificado de BoHV-5, produzindo duas e quatro placas, respectivamente. Nas condições experimentais testadas neste estudo os resultados indicam que as células TT poderiam ser boas candidatas para serem utilizadas para a transfecção de DNA de BoHV-5.

The conditions for an efficacious transfection by calcium phosphate method can vary substantially. In this study the most appropriate cell type for transfection of bovine herpesvirus type 5 (BoHV-5) DNA using this technique was tested. It was first evaluated the capacity of BoHV-5 multiplication in three different cell types. For this purpose virus concentration was calculated by tissue culture infectious dose 50% assay (TCID50/mL). BoHV-5 was titrated in kidney cells from African green monkey (VERO), cell clone of pig kidney (PKsC3), primary cell culture of calf testis (TT) and bovine kidney cells (MDBK). In Vero and PK15 cells maximum virus concentration reached 103,3 TCID50/mL, whereas in MDBK cells the titre was 106,8 TCID50/mL, similar to that of TT cells. In order to evaluate the transfection efficacy in these cells, plasmid DNA expressing a reporter gene (EGFP) was used. Results showed 4.5% fluorescent cells for TT, 2.9% for PKsC3, 0.5% for VERO and 0.05% for MDBK cells. When PKsC3 and TT cells were transfected with 0.5 mg of BoHV-5 purified DNA, only TT cells gave positive result with two plaques per well. To investigate the influence of viral DNA concentration, TT cells were then transfected with 0.5 mg and 1.0 mg of purified BoHV-5 DNA producing two and four plaques, respectively. Under experimental conditions tested in this study the results indicate that TT cells could be good candidates to be used for transfection of BoHV-5 DNA.

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus (AU)

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus