Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys.

Triple reassortant (TR) H3N2 influenza viruses have been isolated from turkeys in the United States since 2003. These TR H3N2 virus infections have been associated with drastic declines in egg production in breeder turkeys although co-infection with multiple agents could have been responsible for exacerbating the clinical signs. In this study, we experimentally confirmed that TR H3N2 influenza virus alone can cause drastic reduction/complete cessation of egg production and pathology of the reproductive tract in 26-week-old breeder turkeys. We confirmed high levels of virus replication and abundant distribution of avian specific alpha2,3 sialic acid-galactose receptors in the oviduct of these turkeys. Although 2-6-week-old turkeys are routinely used for pathogenicity and vaccine protection studies, the low levels of viral shedding and asymptomatic infections in this age group often pose difficulty in interpretation of results. Our study shows that breeder turkeys should be used to assess the potential pathogenicity of TR H3N2 viruses and the viral titers and pathology of the oviduct as well as egg production data can be good measures of protection following in vivo challenge in vaccine efficacy studies.

Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses.

Avian influenza viruses (AIVs) are isolated routinely and propagated in specific pathogen free embryonated chicken eggs (ECE) and mammalian origin Madin-Darby Canine Kidney (MDCK) cell line. Continuous avian cell lines offer advantages for propagation of AIVs over MDCK cells because they maintain species specificity, and lower recurring costs compared to ECE. In this study, the characteristics of two avian fibroblast cell lines were evaluated, DF-1 (chicken-origin) and QT-6 (quail-origin), and their ability to support the growth of AIVs (n=19) belonging to nine different hemagglutinin subtypes from a variety of avian species. The replication efficiency of the AIVs in QT-6 and DF-1 cells was comparable to those in primary chicken embryo fibroblast (CEF) and MDCK cells. Receptor distribution analysis demonstrated high prevalence of SA alpha2,3-gal linked receptors in QT-6 and DF-1 cells which support a high growth of AIVs in these cell lines. Furthermore, the QT-6 and DF-1 cells supported high plaque-forming ability of representative highly pathogenic Eurasian H5N1 and H7N1 subtype AIVs. These two avian cell lines, especially QT-6 cells, also showed high transfection efficiency and could be useful for reverse genetics based rescue of AIVs. This study indicates that the DF-1 and QT-6 cell lines may be useful as a substitute for primary CEF and MDCK cells for AIV research in the areas of in vitro host range, molecular pathobiology and molecular genetics.

Development of sandwich elisa for the detection of fowl adenovirus 4 associated with hydropericardium syndrome in experimentally infected chicken.

A sandwich ELISA was standardized to detect fowl adenovirus (FAV) group I antigen in various tissues, namely liver, spleen, bursa, thymus and kidneys of chicks experimentally infected with fowl adenovirus 4 (FAV-4) isolated from cases of inclusion body hepatitis-hydropericardium syndrome (IBH-HPS). The assay was found to be more sensitive and more specific in comparison to an agar gel immunodiffusion (AGID) test, as it could detect FAV antigen below the titer of 20,000 TCID50/ml and below 1.14 microg in 5% (w/v) suspensions of liver tissue. In 2-week-old experimentally infected chicks, the antigens were detectable by ELISA in liver from 3 to 15 days, in thymus from 3 to 7 days, and in kidneys, bursa and spleen from 3 to 10 days post infection (p.i.). Maximum antigen concentration in terms of ELISA absorbance values was detected in liver and kidneys, which could be used as tissues of choice for virus isolation or detection of viral antigens from IBH-HPS cases.

Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis.

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.