Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and experimental infection

Abstract

BackgroundRespiratory syncytial virus (RSV) is a common respiratory pathogen that can cause severe pneumonia. In vivo studies of RSV can be difficult due to variation in viral infection and disease severity in some animal models. Factors that may contribute to the variation are decreases in viral titer due to preparation and storage and method of virus administration. Nebulization is one method of RSV administration that provides even distribution of virus to all lung lobes; however, the exact quantity of the virus killed by nebulization is not defined. To test the hypothesis that sucrose enhances RSV stability and infectivity, a series of in vitro experiments were conducted with RSV strain Memphis 37 stored at varying concentrations (0%, 3%, 5%, 8%, 10%, 15%, and 20%) of sucrose as a possible cryo- and nebulization protectant. The optimal in vitro concentration was then assessed in vivo in a lamb model.MethodsPrior to titering the virus on HEp-2 cells, the various virus solutions were subjected to one freeze-thaw cycle and one nebulization cycle. Forty-eight hours after viral plating, infectious foci were detected and counted using immunofluorescent imaging. Titers were determined after freeze-thaw and after freeze-thaw followed by nebulization, then compared to the stock titers (before freezing) as well as to one another to determine the loss of infectivity. To further test this in vivo, lambs 2 to 3-days-old were infected via nebulization with RSV using inoculate containing either 20% sucrose or no sucrose followed by assessments of infection severity.ResultsNebulization of virus in 0% sucrose resulted in a 0.580 log reduction in infectivity while virus in 20% sucrose exhibited a 0.297 log reduction. In vivo studies demonstrated that 20% sucrose enhanced RSV lesions and antigen distribution.ConclusionsThe data suggests that both nebulization and freeze-thawing of RSV in the absence of sucrose cause unacceptable losses in viral infectivity and that sucrose acts as a RSV protectant in both regards.

Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs

“…Sonicated samples were then centrifuged again at 2500 rpm (∼1260 x g) for 10 minutes and the resulting supernatants were added to each of the corresponding supernatants collected initially. The samples were then diluted with a solution of 60% sucrose, 10% fetal bovine serum (FBS) in PBS to obtain 20% w/v total sucrose concentration, which was shown to be beneficial in preserving virulence post freeze-thaw and post-nebulization 60 . The titer of virus was subsequently determined, after one freeze-thaw cycle at -80°C, in a small portion of the resulting stocks that were aliquoted into cryovials for this purpose using the infectious FFU assay.…”

STREPTOCOCCUS PNEUMONIAESEROTYPE 22F INFECTION IN RESPIRATORY SYNCYTIAL VIRUS INFECTED NEONATAL LAMBS ENHANCES MORBIDITY

“…For viral inoculations, infectious focus forming units (IFFU), where only replication competent virus is detected by antibody in limiting dilution assays, were utilized. Two groups were exposed to nebulized RSV M37 (1.27x107 IFFU/mL), as done previously [29,30], on day 0. One of the RSV infected groups was inoculated intratracheally with 2 ml normal saline as a mock Spn infection (RSV group) using syringe and needle, while the second RSV-infected group was inoculated intratracheally with 2 ml solution containing Spn serotype 22F (2x106 CFU/ml) 3 days post-RSV nebulization (RSV-Spn group).…”

“…This strain is a wild type A RSV isolated from the respiratory secretions of an infant hospitalized for bronchiolitis [32,33]. M37 was grown in HELA cells and stored at -80°C in media containing 20% sucrose [29]. 6 mL of 1.27 x 10 7 IFFU/ mL in media containing 20% sucrose or cell-conditioned mock media (also containing 20% sucrose) was nebulized using PARI LC Sprint™ nebulizers to each lamb over 25-30 minutes resulting in the total inhalation of about 3 mL by each lamb [29].…”

“…M37 was grown in HELA cells and stored at -80°C in media containing 20% sucrose [29]. 6 mL of 1.27 x 10 7 IFFU/ mL in media containing 20% sucrose or cell-conditioned mock media (also containing 20% sucrose) was nebulized using PARI LC Sprint™ nebulizers to each lamb over 25-30 minutes resulting in the total inhalation of about 3 mL by each lamb [29]. Spn serotype 22F was grown overnight at 37°C in Todd Hewitt media containing 2% yeast extract, 50 g/ml of gentamicin, and 10% bovine serum.…”

“…Lung RSV viral and Spn bacterial titers: BALF collected from the right caudal lobe at necropsy by flushing the caudal lobe with 5 mL of cold DMIM and collected back several times as done previously [29,30]. Collected BALF was used to evaluate RSV IFFU (Plaque assay that counts the number of syncial cells formed due to viral infection detected by fluid fluorescent antibody technique).…”