Detection of Human Bocavirus mRNA in Respiratory Secretions Correlates with High Viral Load and Concurrent Diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>108 copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections

Molecular characterization of Brazilian avian pneumovirus isolates: comparison between immunochemiluminescent Southern blot and nested PCR

Avian pneumovirus (APV) causes acute respiratory tract infection both in turkeys (turkey rhinotracheitis) and chickens (swollen head syndrome (SHS)) with sudden onset and rapid spread through the flocks. In this study, an immunochemiluminescent Southern blot RT-PCR assay was employed to detect a F gene transcript of the APV in two European turkey isolates and two Brazilian chicken isolates. Limiting dilution PCR was carried out to compare the sensitivity of immunochemiluminescent Southern blot assay and nested PCR assay (nPCR). The sensitivity and specificity of immunochemiluminescent Southern blot RT-PCR assay were comparable to that of nPCR, and at least 100 fold more sensitive than a single PCR amplification. Sequence analysis of the 175 bp product of the F gene revealed 100% identity with APV sequences described earlier. (C) 1999 Elsevier Science B.V. All rights reserved.79223724

Molecular characterization of Brazilian avian pneumovirus isolates using reverse transcription-polymerase chain reaction, restriction endonuclease analysis and sequencing of a G gene fragment

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was employed to amplify a G gene transcript of the avian pneumovirus (APV) in two European turkey isolates and two Brazilian chicken isolates. The PCR products were digested using restriction endonucleases and the restriction patterns were compared with earlier reported isolate patterns. The same PCR products were cloned into pUC18 and sequenced. Restriction patterns and sequence analyses of the approximately 600 bp products from both Brazilian isolates revealed 99% identity with earlier reported subgroup A APV sequence, suggesting that these isolates belong to this subgroup.28547347

Experimental rabies infection in haematophagous bats Desmodus rotundus.

In order to determine the susceptibility and serum neutralizing antibody response of Desmodus rotundus to rabies virus, bats were inoculated with a virus isolated from a naturally infected haematophagous bat. Bats were divided into four groups of 10 animals each. Dilutions of rabies virus containing 100, 1000, 10,000 and 100,000 MICLD50 (lethal dose 50% for mice inoculated by the intracerebral route) were administrated in the pectoral muscle. The presence of rabies virus was detected in brain and salivary glands by fluorescent antibody, mouse inoculation and RT-PCR. The observed mortality for each virus dose was 0, 20, 20 and 60% respectively. Serum neutralizing antibodies were tested for by the rapid fluorescent focus inhibition test, and antibody titres greater than 0.5 IU/ml were found in 53% of bats 30 days after virus inoculation. Resistance to infection was seen in bats that developed low or no detectable antibody response as well as in bats with high titres. Among the 10 bats that died of rabies, eight showed signs of paralytic rabies and two bats showed no clinical signs

Investigation of Influenza A, West Nile and Newcastle Disease Viruses in Birds from the Pantanal Wetlands of Mato Grosso, Brazil

ABSTRACT The Pantanal is the world's largest wetland biome with a seasonal flood pulse that attracts a great diversity of birds, many of which are migratory. Birds can be natural reservoirs Influenza A, West Nile and Newcastle Disease viruses. However, the occurrence of carriers for these viruses in the Pantanal was not verified yet. The present study evaluated the occurrence of natural infection by Influenza A, WN and ND virus of birds in the municipality of Poconé, a subregion of the Pantanal in the state of Mato Grosso, Brazil. A total of 76 birds belonging to 11 orders and 20 families were captured using mist nets. The most representative order was Passeriformes, followed by the other nine orders, which included Columbiformes, Psittaciformes, Charadriiformes and Anseriformes. The most representative family was Thamnophilidae, with 16 individuals (21.0%), followed by the family Tyrannidae with 10 individuals (7.6%) and the family Furnariidae, with eight individuals (10.5%). The bird species were identified, and cloacal and tracheal swab samples were collected. The samples were subjected to RNA extraction and tested for the presence of the three agents by real-time polymerase chain reaction (qRT-PCR). All the sampled birds were considered healthy, had no clinical sign of infection, and were tested negative for the three viruses. Based on our findings, we can conclude that Influenza, West Nile and Newcastle Disease viruses were absent from the samples in this region of the Pantanal wetlands during the period of this study