Sandra Arenhart

Anticorpos anti-Neospora caninum em bovinos, ovinos e bubalinos no Estado do Rio Grande do Sul

The infection by Neospora caninum is distributed worldwide and has been considered an important cause of abortion in cattle, which are intermediate hosts of the parasite. The present article reports an serological survey of the N.caninum infection in 1024 serum samples of cattle, sheep and water buffalo from 55 herds in 16 counties of the state of Rio Grande do Sul (RS). Antibodies to the agent were detected by ELISA in 11.4% (89/781) bovine samples, in 14.6% (24/164) water buffalo and in 3.2% (2/62) sheep sera. Positive cattle were detected in all tested counties. These results demonstrate that N. caninum infection is widespread among bovine and other ruminants in the state. Taken together with previous clinical and pathological reports, these results are indicative of the importance of the parasite as the etiological agent of reproductive failure in cattle in RS.

Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg(-1) day(-1), im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbits brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

Replicação e excreção viral durante a infecção aguda e após a reativação da latência induzida por dexametasona em bezerros inoculados com os herpesvírus bovinos tipo 1 (BHV-1) e 5 (BHV-5)

The efficiency of the establishment and reactivation of latent infection by bovine herpesviruses types 1 and 5 (BHV-1 and 5) was compared. Calves inoculated intranasally with BHV-1 (SV-265, n=6) or BHV-5 (SV-507, n=6) presented a mild to moderate nasal discharge and shed virus in nasal secretions in titers up to 107.81TCID50/ml (mean tissue culture infectious dose) during an average of 10.5 days (6-15 [BHV-1]) or up to 106.7 TCID50/ml during 15.3 days (13-18 [BHV-5]). Dexamethasone administration (Dx; 0.5mg/kg) at day 60pi resulted in reactivation of the infection in all calves. Virus shedding in nasal secretions was detected in titers up to 105.5TCID50/ml during 6 to 9 days (mean: 7.8) in calves inoculated with BHV-1 and in titers up to 106.1TCID50/ml (3 to 12 days, mean: 7.5) in calves inoculated with BHV-5. These results showed that BHV-1 and BHV-5 were capable of establishing and reactivating the latent infection at comparable levels.

Excreção e transmissão do vírus da diarréia viral bovina por bezerros persistentemente infectados

Persistently infected (PI) calves born to cows infected with non-cytopathic bovine virus diarrhea virus (BVDV) represent the main reservoir of the virus in nature. We herein report an investigation on the patterns of virus shedding and transmission by five PI calves produced experimentally through inoculation of pregnant cows with Brazilian BVDV isolates. Five calves that survived intrauterine infection were born healthy, lacking neutralizing antibodies to BVDV and harboring virus in the blood. After weaning - and following the disappearance of colostral antibodies - PI calves were monitored for virus in serum and body secretions (ocular, oral, nasal and genital) at weekly intervals for up to 150 days. For each animal, the virus titers in serum showed minor variations throughout the collections (with one exception that presented an increase late in infection), yet the titers varied widely among animals (from 102 to 106TCID50/mL). Virus shedding in secretions was detected steadily during all the observation period with minor titer variations for each particular animal. The highest titers were generally detected in nasal and ocular secretions (titers 104 to 106TCID50mL) whereas genital and oral secretions usually contained low amount of virus (102 to 103TCID50mL). To evaluate the kinetics of virus transmission by these animals, one PI was introduced on a group of 10 seronegative calves maintained with a high animal density simulating the conditions of an intensive management. All 10 contact calves seroconverted to BVDV by day 30. Another PI calf was introduced into a 48-head herd kept under a low animal density, extensive grass management. Among these animals, 8/48 (16.6%) seroconverted by day 10, 26/48 (54.1%) by day 40 and 37/48 (77%) were seropositive at day 100, when the monitoring was discontinued. These results show that continuous viremia and virus shedding in high titers in secretions by PI animals assure an efficient and rapid virus transmission to contact animals, being the kinetics of transmission much faster under intensive conditions.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada

This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34 days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280->10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived (27.7%); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2%). In summary, the experimental vaccine induced adequate antibody titers in most animals and the immunological response induced by vaccination was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. Hence, this experimental vaccine may be an attractive alternative for the prevention of reproductive losses associated with BVDV infection.

Aspectos virológicos e clínico-patológicos da infecção genital aguda e latente pelo herpesvírus bovino tipo 1.2 em bezerras infectadas experimentalmente

ABSTRACT.- Henzel A., Diel D.G., Arenhart S., Vogel F.S.F., Weiblen R. & Flores E.F.2008. [ Virological and clinico-pathological features of acute vulvovaginitis and latentinfection by bovine herpesvirus 1.2 in heifers experimentally infected .] Aspectosvirologicos e clinico-patologicos da infeccao genital aguda e latente pelo herpesvirusbovino tipo 1.2 em bezerras experimentalmente infectadas. Pesquisa Veterinaria Brasileira28(3):140-148. Departamento de Medicina Veterinaria Preventiva, Universidade Federalde Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.brVenereal infection of heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2)may result in vulvovaginitis and transient infertility. The acute infection is followed bythe establishment of latent infection which can be periodically reactivated. We hereindescribe the virology and clinico-pathological aspects of acute and recrudescentvulvovaginitis in heifers inoculated with a Brazilian BoHV-1.2 isolate recovered froman outbreak of balanoposthitis. Genital inoculation of isolate SV-56/90 (10

Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas

This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to > 1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates. At day 240 post-vaccination, the animals received a booster administration (IBSP-2: 107.3 TCID50 and SV-253: 106.8 TCID50). Revaccination resulted in a strong anamnestic response in most animals, with increasing antibody titers mainly to BVDV-2. These are promising results towards the future use of these strains in modified-live vaccines for the control of BVDV infection in Brazil .

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.