The cloning of the virus envelope glycoprotein F of canine distemper virus expressed in Pichia pastoris.

Canine distemper virus (CDV) is a pathogen which affects members of the Canidae family, causing an acute, often fatal, systemic disease. CDV is an RNA virus of the family Paramyxoviridae that contains two envelope glycoproteins: F and HA. In this study, we focused on the envelope glycoprotein F as the main target for neutralizing antibodies produced after infection or vaccination. The complete coding region of the protein (60 kDa) was expressed in the methylotrophic yeast Pichia pastoris, obtained in a recombinant form and secreted to the culture medium. Later, to analyze its immunogenicity, the protein was combined with an oily adjuvant and used to inoculate mice. The results provide evidence supporting a potential application of this recombinant protein as a subunit vaccine.

An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris.

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America / Primeira detecção molecular de co-infecção de vírus de abelhas em Bombus atratus assintomática na América do Sul

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

A polinização é essencial para a produção de alimentos e tem como particularidade a conexão entre os ecossistemas naturais com sistemas de produção agrícola. Recentemente, as perdas de espécies de bumblebee em todo o mundo têm sido relatadas. Neste trabalho, amostras de uma exploração comercial de bumblebee da Argentina, com recente história de mortes foram estudadas utilizando uma Multiplex PCR para a detecção de vírus de abelha mais frequentemente detectados na América do Sul. Todas as amostras analisadas foram positivas para as co-infecções com Deformed wing virus, Black queen cell viruses e Sacbrood virus. Este trabalho descreve o primeiro relato de infecção de Bombus atratus com vírus de abelhas. Uma melhor compreensão das infecções virais em bumblebee e da epidemiologia dos vírus poderia ser de grande importância, uma vez que tais abelhas podem servir como reservatório viral, com possível repercussão tanto na produtividade de abelhas melíferas como afetando-as diretamente.

Systemic infection induced by intranasal inoculation of Bovine herpesvirus 1.1 in pregnant and non-pregnant rabbits.

Bovine herpesvirus (BoHV) type 1.1 (BoHV-1.1) causes repeated outbreaks of upper respiratory disease and abortion in cattle. The systemic effects of BoHV-1.1 in rabbits, using intranasal inoculation are reported. Female rabbits were divided into four groups and inoculated with the virus 10 days before mating, and at 15 or 22 days of pregnancy. Studies of the clinical signs, antibody production, virus isolation, and DNA detection as well as histological and immunohistochemical studies were carried out on lungs, kidneys, spleen, placentas, uteri and foetal tissues. All virus-inoculated animals developed respiratory clinical signs and a humoral response. BoHV-1.1 was isolated from nasal swabs and plasma rich in leukocytes, and viral DNA was detected in blood, dead foetuses and placentas. Histopathological lesions were found in the respiratory tract and some placentas and foetuses were immunohistochemically positive. Intranasal inoculation might be useful to study the systemic effects of BoHV-1.1 infection in the rabbit model.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America.

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

Complete genome amplification of equine influenza virus subtype 2.

This work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (H3N8). A ThermoScript reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase was used. This enzyme has demonstrated higher thermal stability and is described as suitable to make long cDNA with a complex secondary structure. The product obtained by this method can be cloned, used in later sequencing reactions or nested-PCR with the purpose of achieving a rapid diagnosis and characterization of the equine influenza virus type A. This detection assay might be a valuable tool for diagnosis and screening of field samples as well as for conducting molecular studies.

Antibody seroprevalences against rabies in dogs vaccinated under field conditions in Bolivia.

Bolivia currently has one of the highest numbers of cases for human and canine rabies and is thus clue to the elimination process. The objective of the present study was to assess antibody seroprevalences against rabies in dogs vaccinated under field conditions and other factors that might influence the success of the on-going rabies control programmes in an endemic area of the disease, Santa Cruz de la Sierra, Bolivia. All 240 study animals, selected using area-stratified random sampling, were investigated in April 2007. Test prevalences were adjusted for the imperfect test characteristics using the Rogan-Gladen estimator (deterministic and stochastic functions) and Bayesian inference. Ninety-four of the tested 240 vaccinated dogs were classified as test-positive for rabies-specific antibodies. With regard to adjusted overall antibody seroprevalence, Bayesian true prevalence estimates (41%, 95% CI: 37-46%) were lower than both of the Rogan-Gladen estimates. The effect of various epidemiological factors on post-vaccination response was also assessed.

Antibody response to an anti-rabies vaccine in a dog population under field conditions in Bolivia.

Rabies remains an important public health issue in Bolivia, South America. Public concern and fears are most focussed on dogs as the source of rabies. The objective of the present study was to assess immunity of an inactivated suckling mouse brain vaccine against canine rabies used for the official vaccination campaigns under field conditions in an endemic area of rabies in Bolivia. A total of 236 vaccinated and 44 unvaccinated dogs in Santa Cruz de la Sierra, selected using stratified random sampling, were investigated in order to obtain owned dog characteristics and antibody titres against rabies in April 2007. The proportion of vaccinated dogs with an antibody titre exceeded the protection threshold value of 0.5 EU/ml was 58% [95% confidence intervals (CI): 52-65], indicating that vaccination is likely to elicit an antibody response (odds ratio 6.3, 95% CI: 1.2-11.5). The range of geometric mean of antibody titre for vaccinated dogs (0.89 EU/ml; 95% CI: 0.75-1.04) was considered to meet the minimal acceptable level indicating an adequate immune response to the vaccine. However, the titre level was not satisfactory in comparison with the results from other field investigations with inactivated tissue culture vaccines. It is recommended for public health authorities to (1) consider modernizing their vaccine manufacturing method because the level of immunity induced by the current vaccine is comparably low, (2) conduct frequent vaccination campaigns to maintain high levels of vaccination coverage, and (3) actively manage the domestic dog population in the study area, which is largely responsible for rabies maintenance.

The first isolation of equine arteritis virus in Argentina.

This paper describes the first isolation of equine arteritis virus (EAV) in Argentina. The virus was isolated from the semen of an imported seropositive stallion held in isolation at a breeding farm in Tandil in the Buenos Aires Province. In addition, viral nucleic acid was detected in seminal plasma using the reverse-transcription polymerase chain reaction. The isolated virus was propagated in cell cultures and confirmed as EAV by indirect immunofluorescence and virus neutralisation, using a serum specific for the reference Bucyrus strain of EAV. As far as the authors are aware, this is the first time that EAV has been isolated in South America. The equine industry is very important for Argentina and international movement of horses is very intensive. This finding may have effects on the international trade of horses and semen from Argentina.

A polymerase chain reaction for detection of equine herpesvirus-1 in routine diagnostic submissions of tissues from aborted foetuses.

Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.

Rapid diagnosis of pseudorabies virus infection in swine tissues using the polymerase chain reaction (PCR).

In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.

Rapid diagnosis of pseudorabies virus infection in swine tissues using the polymerase chain reaction (PCR)

In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.

[Indirect ELISA for the rapid diagnosis of Equine Influenza]. / ELISA indirecto para el diagnóstico rápido de Influenza Equina.

An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100%, respectively. This indirect ELISA is useful as screening test.

ELISA indirecto para el diagnóstico rápido de Influenza Equina / Indirect ELISA for the rapid diagnosis of Equine Influenza

An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100, respectively. This indirect ELISA is useful as screening test.

Characterization of anti-feline CD8 monoclonal antibodies.

We generated three monoclonal antibodies (mAbs) (2D7, 10C7 and 12A3) reactive to the alpha-chain of feline CD8 (fCD8) molecule. Further we showed that reference anti-fCD8 mAbs, FT2, 3.357 and vpg9 recognize the beta-chain, alpha-chain and alphabeta-complex epitope, respectively. Flow cytometric analysis using these mAbs suggested that fCD8alpha(+)beta(-) cells were present in lymphocytes of spleen, but not significantly in those of thymus, lymph nodes and peripheral blood of normal kittens.

Characterization of several pseudorabies viral strains by virus-neutralization test using monoclonal antibodies.

In the present study, five mouse monoclonal antibodies (MAbs) to the pseudorabies virus (PRV) Yamagata-81 strain were produced. The MAbs were used in cross-neutralization tests and cross-indirect enzyme-linked immunosorbent assay (ELISA) against three PRV viral strains isolated in Argentina and another four obtained from the United States, Japan, France, and Sweden. Four of five MAbs needed the presence of complement to produce or enhance neutralization activity. No differences were observed by ELISA. The MAbs showed different neutralizing activity against PRV strains, suggesting phenotypic heterogeneity among them.

Persistence of high virus neutralizing antibody titers in cats experimentally infected with feline immunodeficiency virus.

The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.

Genetic diversity of Argentine isolates of feline immunodeficiency virus.

We report the nucleotide sequence and genetic diversity of part of the envelope (env) gene of four strains of feline immunodeficiency virus (FIV) isolated from Argentine domestic cats. The DNA encoding the V3 to V5 regions of the env gene of the FIV isolates were amplified by PCR, cloned and sequenced. Phylogenetic analysis revealed that the Argentine isolates did not cluster into a single group; one isolate clustered with subtype B FIV isolated in the USA and Japan, whereas the others formed a new cluster of FIV which might represent a prototype sequence for subtype E.

Molecular cloning of the feline CD8 beta-chain.

Human mouse and rat CD8 have been described as being disulphide-linked heterodimers of alpha and beta chains. More recently the chicken alpha and beta chains were described. In the bovine and feline immune system only the z-chain was reported. In this study we have cloned and determined the nucleotide sequence of a cDNA encoding the beta-chain of the feline T-cell surface antigen CD8. Using a nested polymerase chain reaction- (PCR) and two primer pairs designed from the human CD8 beta cDNA nucleotide sequence, we amplified a 430 base pair fragment from a feline thymus cDNA library which was used as a probe for screening the feline library at high stringency. After three rounds of screening, five clones were isolated. A clone, named FTb-6, containing a 3.8 kilobase pair insert was mapped, sequenced and compared with the published sequences of the genes encoding the human, mouse, rat and avian CD8 beta. We have determined the primary structure of the feline CD8 beta. The feline CD8 beta has an open reading frame, 630 nucleotides in length encoding a protein with 210 amino acid residues and its composition showed that the feline molecule is a member of the immunoglobulin gene super family.

The feline herpesvirus type 1 ICP4 down-regulates feline immunodeficiency virus long terminal repeat (LTR)-directed gene expression via the C/EBP site in the LTR.

We investigated effects of feline herpesvirus type 1 (FHV-1) ICP4 on feline immunodeficiency virus (FIV) long terminal repeat (LTR)-directed gene expression by transient transfection assay in Crandell feline kidney cells. We demonstrated that FHV-1 ICP4 significantly stimulates the FIV LTR after introduction of site-specific mutation of the C/EBP site in the LTR, and the C/EBP site is sufficient to confer inhibitory effects by FHV-1 ICP4 on a heterologous promoter. These results indicate that FHV-1 ICP4 possesses both ability to transactivate FIV LTR-directed gene expression and to down-regulate the FIV LTR via the C/EBP site.