Sequencing human rhinoviruses: direct sequencing versus plasmid cloning.

Human rhinoviruses (RV) are associated with the majority of viral respiratory illnesses in infants, children and adults. Over the last several years, researchers have begun to sequence the many different species and strains of RV in order to determine if certain species were associated with increased disease severity. There are a variety of techniques employed to prepare samples for sequencing. One method utilizes plasmid-cloning, which is expensive and takes several hours to complete. Recently, some investigators have instead used direct sequencing to sequence RV strains, allowing for omission of the time- and labor-intensive cloning step. This study formally compares and contrasts the sequencing results obtained from plasmid-cloning and direct Sanger sequencing of a 500 base pair PCR product covering the VP4/VP2 region of RV. A slightly longer sequence (by 65 base pairs on average) was obtained when specimens were plasmid-cloned, and the sequences were 86% similar. After trimming the extra base pairs from the cloned sequences, the sequences were 99.7% identical. Overall success of directly sequencing samples was similar to that of cloning, 5% on average failed for each technique. Therefore, in many instances, directly sequencing samples may be considered in lieu of the more expensive and time-consuming plasmid-cloning technique.

The Argentina Premature Asthma and Respiratory Team (APART): objectives, design, and recruitment results of a prospective cohort study of viruses and wheezing in very low birth weight infants.

BACKGROUND: Asthma and wheezing account for a substantial disease burden around the world. Very low birth weight (VLBW, <1500 grams) infants are at an increased risk for the development of severe acute respiratory illness (ARI) and recurrent wheeze/asthma. The role of respiratory viruses in asthma predisposition in premature infants is not well understood. Preliminary evidence suggests that infection with human rhinovirus (RV) early in life may contribute to greater burden of asthma later in life. METHODS: A prospective cohort study of premature VLBW infants from Buenos Aires, Argentina, was enrolled year-round during a three-year period in the neonatal intensive care unit and followed during every ARI and with monthly well visits during the first year of life. Longitudinal follow-up up until age five years is ongoing. RESULTS: This report describes the objectives, design, and recruitment results of this prospective cohort. Two hundred and five patients were enrolled from August 2011 through January 2014, and follow-up is ongoing. A total of 319 ARI episodes were observed from August 2011 to July 2014, and 910 well visits occurred during this time period. CONCLUSIONS: The Argentina Premature Asthma and Respiratory Team (APART) is a unique cohort consisting of over 200 patients and over 1200 specimens who have been and will continue to be followed intensively from NICU discharge to capture baseline risk factors and every ARI, with interceding well visits during the first year of life, as well as longitudinal follow-up to age 5 years for asthma and atopy outcomes.