Rita de Cássia Nasser Cubel Garcia

Molecular characterization of canine coronavirus strains circulating in Brazil

Abstract To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil. To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.

Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

Canine parvovirus infection in puppies with gastroenteritis in Niterói, Rio de Janeiro, Brazil from 1995 to 1997.

Amostras fecais de caes com gastrenterite, ate 6 meses de idade, foram testadas para a presenca do parvovirus canino (CPV-2) pela reacao de hemaglutinacao (HA) e confirmadas pela reacao de inibicao da hemaglutinacao (HI). Quarenta das 79 amostras, recebidas no periodo de abril de 1995 a junho de 1997, foram consideradas positivas. Aproximadamente 70% destas amostras foram obtidas de animais entre 2 e 4 meses de idade, epoca em que o risco de contrairem a infeccao pelo CPV-2 e alto apesar da vacinacao. Nenhuma variacao sazonal da infeccao pelo parvovirus canino pode ser observada, e um estudo retrospectivo realizado na PolVet - UFF mostrou que em um periodo de 6 anos (1991-97), casos de gastrenterite ocorreram durante todos os anos em Niteroi, sem uma sazonalidade definida.

Clinical features and laboratory findings of human parvovirus B19 in human immunodeficiency virus-infected patients

Immunocompromised patients may develop severe chronic anaemia when infected by human parvovirus B19 (B19V). However, this is not the case in human immunodeficiency virus (HIV)-infected patients with good adherence to highly active antiretroviral treatment (HAART). In this study, we investigated the clinical evolution of five HIV-infected patients receiving HAART who had B19V infections confirmed by serum polymerase chain reaction. Four of the patients were infected with genotype 1a strains and the remaining patient was infected with a genotype 3b strain. Anaemia was detected in three of the patients, but all patients recovered without requiring immunoglobulin and/or blood transfusions. In all cases, the attending physicians did not suspect the B19V infections. There was no apparent relationship between the infecting genotype and the clinical course. In the HAART era, B19V infections in HIV-positive patients may be limited, subtle or unapparent.

Characterization of parvoviruses from domestic cats in Brazil

To characterize Feline parvovirus (FPV) circulating in domestic cats in Brazil, 51 fecal samples from unvaccinated domestic cats were collected during 2004–2005. Six parvoviruses were characterized by hemagglutination (HA) assay at different pH values and temperatures and by polymerase chain reaction (PCR) using different pairs of primers. However, data obtained from HA and PCR did not allow the discrimination between FPV and Canine parvovirus (CPV). Two regions of the VP2 capsid gene (1,171-bp fragment) involved in controlling canine and feline host range were sequenced; 9 synonymous and 10 non-synonymous nucleotide substitutions were detected. All samples were confirmed as FPV by nucleotide sequencing, but 3 feline samples had amino acid changes at residues 93, 375, and 426, which are present in canine strains. The phylogenetic tree built based on nucleotide sequences showed that Brazilian feline samples form a cluster distinct from other parvoviruses deposited in GenBank. Taken together, the findings reinforce the importance of monitoring the continuous evolution of CPV and FPV in the feline population in Brazil.

Molecular diversity of human parvovirus B19 during two outbreaks of erythema infectiosum in Brazil

This study was conducted to provide information on the genetic diversity of human parvovirus B19 (B19V) circulating in the municipality of Niterói, Rio de Janeiro, Southeast Brazil during 1996-2006, a period with two distinct outbreaks of B19V infection: 1999-2000 and 2004-2005. A total of 27 sera from patients with erythema infectiosum and five sera from HIV-infected patients that tested positive for B19V DNA during the study period were analyzed. To genotype B19V strains, a semi-nested PCR for partial amplification of the capsid gene was performed and sequence analysis revealed that 31 sequences belonged to subgenotype 1a (G1a) of the main genotype 1 and one sequence was characterized as subgenotype 3b (G3b). The phylogenetic tree supported the division of the G1a into two well-defined clades with 1.3% of divergence. The low diversity of the G1a strains may be explained by the fact that all patients had acute B19V infection and 30/32 sera were collected during two distinct outbreaks. The G3b strain was from an HIV-infected patient who seroconverted to anti-B19 IgG antibodies in September/2005. This is the first report of G3b in the state of Rio de Janeiro.

Detection and molecular characterization of caliciviruses (vesivirus and norovirus) in an outbreak of acute diarrhea in kittens from Brazil.

Abstract Feline caliciviruses (FCVs) have occasionally been described in cats in association with enteric disease, but an etiological role for these viruses in acute gastroenteritis is still unclear. In this study, molecular characterization of FCV and feline norovirus (FNoV) was undertaken using real-time PCR (RT-PCR) and sequence analysis of the ORF1 region in fecal specimens from 29 diarrheic cats. The specimens were also screened for parvovirus, coronavirus, astrovirus and group A rotavirus. A quantitative one step RT-PCR was also performed to detect and quantitate NoV genogroup IV and the role of these animal caliciviruses in feline gastroenteritis was investigated. This is the first description of enteric FCV and FNoV in South America.

Parvovirus B19 seroconversion in a cohort of human immunodeficiency virus-infected patients

Erythrovirus B19 (B19V) infection may cause red cell aplasia in patients infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has improved the immune function of these patients by modifying the course of B19V infection. The purpose of this study was to estimate the frequency of B19 seroconversion in a cohort of HIV-infected patients and evaluate the occurrence of B19V-related anaemia during the seroconversion period. Adult HIV-infected patients were studied at a public hospital in Niterói, state of Rio de Janeiro, Brazil. IgG and IgM antibodies against B19V were detected by an enzyme-linked immunosorbent assay and B19 viraemia was assayed by polymerase chain reaction. Medical records were reviewed for any clinical evaluation of anaemia. Seroconversion was detected in 31.8% of the 88 individuals who began the study as anti-B19V IgG-negative. No clinical manifestations of B19V infection were detected during the period of seroconversion. Patients who seroconverted were 5.40 times more likely to have anaemia than those who did not [odds ratio 5.40 (95% confidence interval: 1.33-22.93)]. Anaemia was detected in eight patients. All patients recovered from anaemia by either beginning or continuing HAART, without requiring blood transfusions. In the HAART era, B19V infection may only be associated with a course of disease characterised by less severe chronic anaemia. This milder course of B19V-associated disease is likely due to the increased immune function of HAART-treated patients.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Parvovirus B19 Infection in a Fatal Case of Acute Liver Failure

B19V has been proposed as an etiologic agent for hepatitis, mainly in children, but this is a rare clinical occurrence. In this article, we report a case of non-A-E acute liver failure in an immunocompetent child with B19 infection. The clinical findings of severe anemia and pancytopenia combined with the detection of anti-B19 Immunoglobulin G (IgG), B19 DNA and B19 mRNA in liver indicate a persistent infection and suggest a diagnosis of parvovirus B19-associated acute liver failure.