Improving Pneumovirus Isolation Using a Centrifugation and AZD1480 Combined Method.

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.

Comparison of the Seeplex reverse transcription PCR assay with the R-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses.

This study evaluated the clinical usefulness of a newly introduced multiplex reverse transcription PCR assay (Seeplex RV; Seegene, Inc., Seoul, Korea) in patients with respiratory symptoms. Fifty clinical respiratory specimens (45 from children, 5 from adults) were tested for 8 viruses (influenza virus type A and B, parainfluenza virus type 1, 2, 3, respiratory syncytial virus type A and B, and adenovirus) by Seeplex RV (S-RV) and R-mix viral culture with immunofluorescence (VC-IF). Forty (80%) of the 50 samples showed concordant results between S-RV and VC-IF; 24 of these showed the same positive and 16 showed the same negative results. Among the 10 discrepant samples, 9 were S-RV-positive and VC-IF-negative. Six were obtained in patients with lower respiratory tract infection. Only 1 sample was VC-IF-positive and S-RV-negative. This patient had pneumonia. In 3 cases, more than 1 virus was identified by S-RV. The total running time of S-RV was 6 hr, which shortens the detection time for the viral presence by 2 workdays compared to VC-IF. In conclusion, S-RV is reliable, rapid, relatively easy to perform, and able to detect more than 1 virus simultaneously. Therefore, implementation of the S-RV assay in clinical laboratories will aid rapid diagnosis and treatment of major viral infections of the respiratory tract.

Susceptibility of ducks to avian pneumovirus of turkey origin.

OBJECTIVE: To determine the susceptibility of ducks to avian pneumovirus (APV) of turkey origin. ANIMALS: 30 Pekin ducks that were 2 weeks old. PROCEDURE: Ducks were assigned to 3 groups (10 ducks/group). Ducks of groups 1 and 2 were inoculated (day 0) with 200 microl of cell-culture fluid containing APV of turkey origin (10(5.5) median tissue-culture infective dose/ml) by the oculonasal (group 1) or oral (group 2) route. Ducks of group 3 served as noninoculated control birds. Two ducks from each group were euthanatized 3, 6, 9, 15, and 21 days after inoculation. Blood samples, tissue samples from the lungs, trachea, nasal turbinates, duodenum, diverticulum vitellinum (Meckel's diverticulum), and cecum, and swab specimens from the choana, cloaca, and trachea were obtained from all birds during necropsy and examined for APV by use of reverse transcriptase-polymerase chain reaction (RT-PCR), virus isolation, and histologic examination. Blood samples also were examined for APV antibodies, using an ELISA. RESULTS: Tissue samples obtained up to 21 days after inoculation had positive results when tested by use of RT-PCR. Virus was isolated from nasal turbinates of birds inoculated via the oculonasal route. Serum samples obtained 15 and 21 days after inoculation had positive results when tested for APV-specific antibody. Clinical signs of disease were not observed in ducks inoculated with APV of turkey origin. CONCLUSIONS AND CLINICAL RELEVANCE: Ducks inoculated with APV of turkey origin may not develop clinical signs of disease, but they are suspected to play a role as nonclinical carriers of APV.

Ultrastructural study of turkey rhinotracheitis virus infection in turbinates of experimentally infected chickens.

Ultrastructural changes associated with turkey rhinotracheitis virus infection were studied in turbinates of chickens experimentally infected with the isolate CVL 14/86/1. Chickens were sacrificed at 3, 5 and 7 days after inoculation and samples of the middle turbinate were taken, fixed, dehydrated and embedded in an hydrophilic resin. An immunofluorescence technique on semithin sections was carried out and viral antigen was observed in the cytoplasm and associated to cilia of the turbinate epithelial cells, on days 3 and 5 after inoculation. Ultrastructurally, gold stained intracytoplasmic nucleocapsid aggregates of turkey rhinotracheitis virus were observed in ciliated and non-ciliated epithelial cells, as well as budding virus particles, at days 3 and 5 postinoculation. Different ultrastructural abnormalities, including cytoplasmic blebs, clumping and loss of cilia were observed in the apical cell membrane of many infected cells, associated with the presence of intracytoplasmic inclusions. On day 5 after inoculation, substitution of ciliated and non-ciliated epithelial cells was noted and many desquamated epithelial cells were observed within the lumina. Regenerative changes in the ciliated epithelium were observed by day 7 postinoculation. These results indicate that turkey rhinotracheitis virus is able to replicate in ciliated and non-ciliated epithelial cells causing severe alterations to the cell surface and ciliary apparatus of the turbinate epithelium. Viral-induced damage to the turbinate epithelium could enhance the susceptibility of epithelial cells to secondary bacterial infection.