Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil

Abstract: Canid herpesvirus 1 (CHV-1) is a widespread pathogen of dogs and produces infertility, abortions and severe systemic disease in young puppies. Clinical data indicate the circulation of CHV-1 among Brazilian dogs yet definitive diagnosis has rarely been accomplished. This article describes the clinicopathological findings of four independent cases/outbreaks of neonatal disease by CHV-1 in Bulldog puppies followed by virus identification and genetic characterization. Three events occurred in a kennel holding dogs of different breeds at reproductive age (March 2013, October 2013 and April 2014). Puppies from three French or English Bulldog litters, aging 9 to 30 days were affected, presenting dyspnea, agonic breathing, pale mucous, abdominal pain and tension, evolving to death within about 24 hours. At necropsy, the puppies presented necrohemorrhagic hepatitis, multifocal and moderate necrohemorrhagic nephritis and fibrinonecrotic interstitial pneumonia. Virus isolation was positive in clinical specimens from one litter and CHV-1 DNA was detected by PCR in tissues from all four cases. Virus-neutralizing assays with samples of the affected kennel revealed 9/12 adult animals with high antibody titers to CHV-1. Nucleotide sequencing of glycoprotein B, C and D genes revealed 99-100% of identity among the viruses and with CHV-1 sequences available in GenBank. Phylogenetic analyses of gC sequences showed a segregation of the samples, even among three isolates from the same kennel. These findings support CHV-1 infection as the cause of disease and death in these dog litters, reinforcing the need for correct etiologic diagnosis, prevention and immunization against CHV-1 in dogs from Southern Brazil.

Further molecular characterization of weed-associated begomoviruses in Brazil with an emphasis on Sida spp

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often associated with weed plants. The aim of this study was to further characterize the diversity of begomoviruses infecting weeds (mostly Sida spp.) in Brazil. Total DNA was extracted from weed samples collected in Viçosa (Minas Gerais state) and in some municipalities of Alagoas state in 2009 and 2010. Viral genomes were amplified by RCA, cloned and sequenced. A total of 26 DNA-A clones were obtained. Sequence analysis indicated the presence of 10 begomoviruses. All viral isolates from Blainvillea rhomboidea belonged to the same species, Blainvillea yellow spot virus (BlYSV ), thereby suggesting that BlYSV may be the only begomovirus present in this weed species. Four isolates represent new species, for which the following names are proposed: Sida yellow blotch virus (SiYBV), Sida yellow net virus (SiYNV), Sida mottle Alagoas virus (SiMoAV) and Sida yellow mosaic Alagoas virus (SiYMAV). Recombination events were detected among the SiYBV isolates and in the SiYNV isolate. These results constitute further evidence of the high species diversity of begomoviruses in Sida spp. However, the role of this weed species as a source of begomoviruses infecting crop plants remains to be determined.

Production of recombinant antigens in plants for animal and human immunization - a review

Plants present a cost effective production system for high value proteins. There is an increasing world demand for cheap vaccines that can be readily administered to the population, especially in economically less developed regions. A promising concept is the production of vaccines in plants that could be grown locally. Expression of antigenic peptides in the palatable parts of plants can lead to the production of edible active vaccines. Two major strategies are: i) to express antigens in transgenic plants, and ii) to produce antigenic peptides on the surface of plant viruses that could be used to infect host plants. This review considers the experimental data and early results for both strategies, and discusses the potential and problems of this new technology

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

Relationship of cytokines and cytokine signaling to immunodeficiency disorders in the mouse

The contributions of cytokines to the development and progression of disease in a mouse model of retrovirus-induced immunodeficiency (MAIDS) are controversial. Some studies have indicated an etiologic role for type 2 cytokines, while others have emphasized the importance of type 1 cytokines. We have used mice deficient in expression of IL-4, IL-10, IL-4 and IL-10, IFN-ACE="Symbol">g, or ICSBP - a transcriptional protein involved in IFN signaling - to examine their contributions to this disorder. Our results demonstrate that expression of type 2 cytokines is an epiphenomenon of infection and that IFN-ACE="Symbol">g is a driving force in disease progression. In addition, exogenously administered IL-12 prevents many manifestations of disease while blocking retrovirus expression. Interruption of the IFN signaling pathways in ICSBP-/- mice blocks induction of MAIDS. Predictably, ICSBP-deficient mice exhibit impaired responses to challenge with several other viruses. This immunodeficiency is associated with impaired production of IFN-ACE="Symbol">g and IL-12. Unexpectedly, however, the ICSBP-/- mice also develop a syndrome with many similarities to chronic myelogenous leukemia in humans. The chronic phase of this disease is followed by a fatal blast crisis characterized by clonal expansions of undifferentiated cells. ICSBP is thus an important determinant of hematopoietic growth and differentiation as well as a prominent signaling molecule for IFNs

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Regulation of transgene expression in genetic immunization

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-ACE="Symbol">g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-ACE="Symbol">g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-ACE="Symbol">g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Prevalence and risk factors for HBV, HCV and HDV infections among injecting drug users from Rio de Janeiro, Brazil

Viral hepatitis constitutes a major health issue, with high prevalence among injecting drug users (IDUs). The present study assessed the prevalence and risk determinants for hepatitis B, C and D viruses (HBV, HCV and HDV) infections among 102 IDUs from Rio de Janeiro, Brazil. Serological markers and HCV-RNA were detected by enzyme immunoassay and nested PCR, respectively. HCV genotyping was determined by restriction fragment length polymorphism analysis (RFLP). HBsAg, anti-HBc and anti-HBs were found in 7.8, 55.8 and 24.7% of IDUs, respectively. In the final logistic regression, HBV infection was independently associated with male homosexual intercourse within the last 5 years (odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.8). No subject presented anti-delta (anti-HD). Anti-HCV was detected in 69.6% of subjects, and was found to be independently associated with needle sharing in the last 6 months (OR 3.4; 95% CI 1.3-9.2) and with longer duration of iv drug use (OR 3.1; 95% CI 1.1-8.7). These data demonstrate that this population is at high risk for both HBV and HCV infection. Among IDUs from Rio de Janeiro, unprotected sexual intercourse seems to be more closely associated with HBV infection, whereas HCV is positively correlated with high risk injecting behavior. Comprehensive public health interventions targeting this population and their sexual partners must be encouraged.

Sloth biology: an update on their physiological ecology, behavior and role as vectors of arthropods and arboviruses

This is a review of the research undertaken since 1971 on the behavior and physiological ecology of sloths. The animals exhibit numerous fascinating features. Sloth hair is extremely specialized for a wet tropical environment and contains symbiotic algae. Activity shows circadian and seasonal variation. Nutrients derived from the food, particularly in Bradypus, only barely match the requirements for energy expenditure. Sloths are hosts to a fascinating array of commensal and parasitic arthropods and are carriers of various arthropod-borne viruses. Sloths are known reservoirs of the flagellate protozoan which causes leishmaniasis in humans, and may also carry trypanosomes and the protozoan Pneumocystis carinii.

HIV-1 subtypes among intravenous drug users from two neighboring cities in São Paulo State, Brazil

In order to assess the molecular epidemiology of HIV-1 in two neighboring cities located near the epicenter of the HIV-1 epidemics in Brazil (Santos and São Paulo), we investigated 83 HIV-1 strains obtained from samples collected in 1995 from intravenous drug users. The V3 through V5 region of the envelope of gp 120 was analyzed by heteroduplex mobility analysis. Of the 95 samples, 12 (12.6%) were PCR negative (6 samples from each group); low DNA concentration was the reason for non-amplification in half of these cases. Of the 42 typed cases from São Paulo, 34 (81%, 95% confidence limits 74.9 to 87.0%) were B and 8 (19%, 95% confidence limits 12.9 to 25.0%) were F, whereas of the 41 typed cases from Santos, 39 (95%, 95% confidence limits 91.6 to 98.4%) were B and 2 (5%, 95% confidence limits 1.6 to 8.4%) were C. We therefore confirm the relationship between clade F and intravenous drug use in São Paulo, and the presence of clade C in Santos. The fact that different genetic subtypes of HIV-1 are co-circulating indicates a need for continuous surveillance for these subtypes as well as for recombinant viruses in Brazil.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Immune response induced in mice oral immunization with cowpea severe mosaic virus

There is increasing interest in the immune response induced by plant viruses since these could be used as antigen-expressing systems in vaccination procedures. Cowpea severe mosaic virus (CPSMV), as a purified preparation (300 g of leaves, 2 weeks post-inoculation), or crude extract from cowpea (Vigna unguiculata) leaves infected with CPSMV both administered by gavage to Swiss mice induced a humoral immune response. Groups of 10 Swiss mice (2-month-old females) were immunized orally with 10 daily doses of either 50 µg viral capsid protein (boosters of 50 µg at days 21 and 35 after immunization) or 0.6 mg protein of the crude extract (boosters of 0.6 mg at days 21 and 35 after immunization). Anti-CPSMV antibodies were quantified by ELISA in pooled sera diluted at least 1:400 at days 7, 14, 21, 28, 35 and 42 after the 10th dose. IgG and IgA against CPSMV were produced systemically, but IgE was not detected. No synthesis of specific antibodies against the proteins of leaf extracts from V. unguiculata, infected or not with CPSMV, was detected. The use of CPSMV, a plant-infecting virus that apparently does not induce a pathogenic response in animals, induced a humoral and persistent (at least 6 months) immune response through the administration of low antigen doses by gavage. These results raise the possibility of using CPSMV either as a vector for the production of vaccines against animal pathogens or in quick and easy methods to produce specific antisera for viral diagnosis.