Identification and characterization of a Hydrangea ringspot virus isolate infecting hydrangea in São Paulo State

Hydrangea plants showing leaves with chlorotic and necrotic rings from Arujá Municipality, São Paulo State, were analyzed for the identification of the viral species. Elongated filamentous particles of 490 nm were visualized under transmission electron microscope. Oligonucleotides for Hydrangea ringspot virus (HdRSV), a potexvirus commonly found in Europe and in the United States, were tested using total RNA from hydrangea plants, amplifying two fragments, one around 550 and another one of 250 nucleotides. Nucleotide identity with HdRSV (accession number AJ 707100.1) was 96% and 88% for the longest and shortest fragment, respectively, indicating the presence of this virus. To evaluate its dissemination in the matrices of hydrangea used in the commercial production, 17 samples were collected in the region of Arujá, and eight were infected by HdRSV. For the analyzed viral replicase portion, the isolates from the varieties 'Azul LZR', 'Rosita', 'Renat Blue' and 'Vermelho Comum' did not differ in their amino acid sequences from isolates with sequences deposited in the GenBank (accession numbers AY 707100 and NC_006943). The isolates from 'Azul Rendado' and "Rosa Japonesa' showed few differences but were related to the remaining isolates. An antiserum was obtained for HdRSV and can be efficiently used to detect such virus in hydrangea and Primula malacoides, another ornamental plant also infected by HdRSV.

Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5% of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

Hepatitis C virus infection among Brazilian hemophiliacs: a virological, clinical and epidemiological study

We determined and analyzed risk factors of hepatitis C virus (HCV)-infected Brazilian hemophiliacs according to their virological, clinical and epidemiological characteristics. A cross-sectional and retrospective study of 469 hemophiliacs was carried out at a Brazilian blood center starting in October 1997. The prevalence of HCV infection, HCV genotypes and factors associated with HCV RNA detection was determined. The seroprevalence of anti-HCV antibodies (ELISA-3.0) was 44.6% (209/469). Virological, clinical and epidemiological assessments were completed for 162 positive patients. There were seven (4.3%) anti-HCV seroconversions between October 1992 and October 1997. During the same period, 40.8% of the positive anti-HCV hemophiliacs had abnormal alanine transaminase (ALT) levels. Plasma HCV RNA was detected by nested-RT-PCR in 116 patients (71.6%). RFLP analysis showed the following genotype distribution: HCV-1 in 98 hemophiliacs (84.5%), HCV-3 in ten (8.6%), HCV-4 in three (2.6%), HCV-2 in one (0.9%), and not typeable in four cases (3.4%). Univariate analysis indicated that older age (P = 0.017) and abnormal ALT levels (P = 0.010) were associated with HCV viremia, while the presence of inhibitor antibodies (P = 0.024) and HBsAg (P = 0.007) represented a protective factor against the presence of HCV RNA. These findings may contribute to a better understanding of the relationship between HCV infection and hemophilia.

Characterization of bovine respiratory syncytial virus isolated in Brazil

This paper presents the first isolation of bovine respiratory syncytial virus in Brazil and its physicochemical, morphological and molecular characterization. The virus was isolated from 33 samples of nasotracheal secretions, successively inoculated into a Madin-Darby bovine kidney cell culture, which was characterized by physicochemical tests and morphological observation by electron microscopy. The Brazilian sample is an RNA pleomorphic, enveloped, thermolabile and non-hemagglutinating spicular virus. Reverse transcription, followed by nested polymerase chain reaction (nRT-PCR) assay was carried out using oligonucleotides B1, B2A, B3 and B4 for the fusion proteins (F) and B5A, B6A, B7A and B8 for the attachment protein (G). The nRT-PCR-F amplified a fragment of 481 bp corresponding to part of the gene that codes for protein F, whereas nRT-PCR-G amplified a fragment of 371 bp, in agreement with part of the G gene. The virus isolated from Brazilian samples in this study corresponded to the bovine respiratory syncytial virus, and RT-PCR proved to be useful for the diagnosis of bovine clinical samples.

Hepatitis C virus infection in a Brazilian population with sickle-cell anemia

Patients with sickle-cell anemia submitted to frequent blood transfusions are at risk of contamination with hepatitis C virus (HCV). Determination of HCV RNA and genotype characterization are parameters that are relevant for the treatment of the viral infection. The objective of the present study was to determine the frequency of HCV infection and the positivity for HCV RNA and to identify the HCV genotype in patients with sickle-cell anemia with a history of blood transfusion who had been treated at the Hospital of the HEMOPE Foundation. Sera from 291 patients were tested for anti-HCV antibodies by ELISA 3.0 and RIBA 3.0 Chiron and for the presence of HCV RNA by RT-PCR. HCV genotyping was performed in 19 serum samples. Forty-one of 291 patients (14.1%) were anti-HCV positive by ELISA and RIBA. Both univariate and multivariate analysis showed a greater risk of anti-HCV positivity in those who had started a transfusion regime before 1992 and received more than 10 units of blood. Thirty-four of the anti-HCV-positive patients (34/41, 82.9%) were also HCV RNA positive. Univariate analysis, used to compare HCV RNA-negative and -positive patients, did not indicate a higher risk of HCV RNA positivity for any of the variables evaluated. The genotypes identified were 1b (63%), 1a (21%) and 3a (16%). A high prevalence of HCV infection was observed in our patients with sickle-cell anemia (14.1%) compared to the population in general (3%). In the literature, the frequency of HCV infection in sickle-cell anemia ranges from 2 to 30%. The serological screening for anti-HCV at blood banks after 1992 has contributed to a better control of the dissemination of HCV infection. Because of the predominance of genotype 1, these patients belong to a group requiring special treatment, with a probable indication of new therapeutic options against HCV.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52%) and cirrhosis in 11/52 (21%) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Genetic variability of hepatitis A virus isolates in Rio de Janeiro: implications for the vaccination of school children

The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5%. HAV-RNA was detected in 46% IgM-positive serum samples and in 16% stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90% of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

Geographic distribution of hepatitis C virus genotypes in Brazil

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV- RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9% (1,095) for genotype 1, 4.6% (78) for genotype 2, 30.2% (510) for genotype 3, 0.2% (3) for genotype 4, and 0.1% (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1%), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4%), especially in Mato Grosso State (25.8%), while genotype 3 was more common in the South (43.2%). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Hepatitis C virus seroprevalence and genotypes in patients with diffuse connective tissue diseases and spondyloarthropathies

Many extrahepatic manifestations, including rheumatic diseases, have been reported to be associated with hepatitis C virus (HCV) infection. In order to investigate the prevalence of HCV infection among patients with rheumatic diseases, in the present study we interviewed 367 patients and tested their blood samples for HCV antibodies (anti-HCV) by an enzyme-linked immunosorbent assay. Anti-HCV-reactive samples were retested for confirmation by a line immunoassay and also for HCV RNA detection by the polymerase chain reaction. HCV RNA-positive samples were genotyped by INNO-LIPA. An overall HCV infection prevalence of 1.9% (7/367) was found. Of the 7 HCV-infected patients, 4 had systemic lupus erythematosus and 3 rheumatoid arthritis, resulting in positivity rates of 2.3 and 3.4%, respectively. HCV RNA genotyping revealed the presence of subtypes 1a (57.1%), 1b (28.6%) and 3a (14.3%). The clinical course was favorable for all HCV-infected patients, except one, who died due to renal insufficiency related to lupus nephritis. These results demonstrate a low HCV infection prevalence among the population studied. In the few positive cases, we observed no adverse influence of this infection on the clinical evolution of the rheumatic disease.

Hepatitis C virus infection, cryoglobulinemia, and peripheral neuropathy: a case report

Hepatitis C virus (HCV) is essentially hepatotropic but its manifestations can extend beyond the liver. It can be associated with autoimmune diseases, such as mixed cryoglobulinemia, membranoproliferative glomerulonephritis, autoimmune thyroiditis, and lymphoproliferative disorders. The mechanisms that trigger these manifestations are not completely understood. We describe a 48-year-old man with chronic HCV infection (circulating HCV RNA and moderate hepatitis as indicated by liver biopsy), cryoglobulinemia, and sensory and motor peripheral neuropathy. The diagnosis of multineuropathy was confirmed by clinical examination and electromyographic tests. A nerve biopsy revealed an inflammatory infiltrate in the perineurial space and signs of demyelination and axonal degeneration. The patient had no improvement of neurological symptoms with the use of analgesics and neuro-modulators. He was then treated with interferon-alpha (3 million units subcutaneously, 3 times per week) and ribavirin (500 mg orally, twice a day) for 48 weeks. Six months after the end of therapy, the patient had sustained viral response (negative HCV RNA) and remission of neurological symptoms, but cryoglobulins remained positive. A review of the literature on the pathogenesis and treatment of neurological manifestations associated with HCV infection is presented. This report underscores the need for a thorough evaluation of HCV-infected patients because of the possibility of extrahepatic manifestations. Antiviral treatment with interferon and ribavirin can be effective and should be considered in patients with neurological complications associated with HCV infection.

Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.