In Vitro and in Vivo Stimulation of Murine Lymphocytes by Human Respiratory Syncytial Virus Strains

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

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Mitoxantrone Shows In Vitro, but Not In Vivo Antiviral Activity against Human Respiratory Syncytial Virus

Biomedicines ◽

10.3390/biomedicines9091176 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1176

Author(s):

Patricia G. de la Sota ◽

Elena Lorente ◽

Laura Notario ◽

Carmen Mir ◽

Oscar Zaragoza ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Infections ◽

The Elderly ◽

Human Respiratory Syncytial Virus ◽

Mice Model ◽

Drug Candidates ◽

Antiviral Activities ◽

Syncytial Virus

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, this virus poses a serious health risk in immunocompromised individuals and the elderly. HRSV is also a major nosocomial hazard in healthcare service units for patients of all ages. Therefore, the development of antiviral treatments against HRSV is a global health priority. In this study, mitoxantrone, a synthetic anthraquinone with previously reported in vitro antiprotozoal and antiviral activities, inhibits HRSV replication in vitro, but not in vivo in a mice model. These results have implications for preclinical studies of some drug candidates.

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Genetic Stability of Parainfluenza Virus 5-Vectored Human Respiratory Syncytial Virus Vaccine Candidates after In Vitro and In Vivo Passage

Journal of Virology ◽

10.1128/jvi.00559-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 6

Author(s):

Shannon I. Phan ◽

Carolyn M. Adam ◽

Zhenhai Chen ◽

Michael Citron ◽

Xiaoping Liang ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Genetic Stability ◽

Human Respiratory Syncytial Virus ◽

Parainfluenza Virus ◽

Vaccine Candidates ◽

Consensus Sequences ◽

Parainfluenza Virus 5 ◽

Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines. IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo. We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

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Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

PLoS ONE ◽

10.1371/journal.pone.0127098 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0127098 ◽

Cited By ~ 23

Author(s):

D. Tien Nguyen ◽

Rogier Louwen ◽

Karin Elberse ◽

Geert van Amerongen ◽

Selma Yüksel ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Streptococcus Pneumoniae ◽

Virus Infection ◽

Respiratory Syncytial Virus Infection ◽

Human Respiratory Syncytial Virus ◽

Syncytial Virus

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Inhibitory Effect of Sargassum fusiforme and Its Components on Replication of Respiratory Syncytial Virus In Vitro and In Vivo

Viruses ◽

10.3390/v13040548 ◽

2021 ◽

Vol 13 (4) ◽

pp. 548

Author(s):

Kiramage Chathuranga ◽

Asela Weerawardhana ◽

Niranjan Dodantenna ◽

Lakmal Ranathunga ◽

Won-Kyung Cho ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Antiviral Agent ◽

Active Components ◽

Sargassum Fusiforme ◽

Antiviral Effects ◽

Improve Life Expectancy ◽

Rsv Infection ◽

Syncytial Virus

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

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In Vitro and In Vivo Fitness of Respiratory Syncytial Virus Monoclonal Antibody Escape Mutants

Journal of Virology ◽

10.1128/jvi.01387-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11651-11657 ◽

Cited By ~ 20

Author(s):

Xiaodong Zhao ◽

Enmei Liu ◽

Fu-Ping Chen ◽

Wayne M. Sullender

Keyword(s):

Cell Culture ◽

Monoclonal Antibody ◽

Respiratory Syncytial Virus ◽

Viral Population ◽

Nucleotide Substitutions ◽

Cotton Rats ◽

Syncytial Virus ◽

In Vivo Growth

ABSTRACT Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.

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Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats

Journal of General Virology ◽

10.1099/jgv.0.001471 ◽

2020 ◽

Vol 101 (10) ◽

pp. 1056-1068

Author(s):

Linda J. Rennick ◽

Sham Nambulli ◽

Ken Lemon ◽

Grace Y. Olinger ◽

Nicholas A. Crossland ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Tract ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Human Respiratory Syncytial Virus ◽

Subtype B ◽

Cotton Rats ◽

Syncytial Virus ◽

Subtype A

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

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Exacerbation of Influenza A Virus Disease Severity by Respiratory Syncytial Virus Co-Infection in a Mouse Model

Viruses ◽

10.3390/v13081630 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1630 ◽

Cited By ~ 1

Author(s):

Junu A. George ◽

Shaikha H. AlShamsi ◽

Maryam H. Alhammadi ◽

Ahmed R. Alsuwaidi

Keyword(s):

T Cells ◽

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Influenza A ◽

Immune Cell ◽

Virus Disease ◽

Viral Loads ◽

Syncytial Virus

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are leading causes of childhood infections. RSV and influenza are competitive in vitro. In this study, the in vivo effects of RSV and IAV co-infection were investigated. Mice were intranasally inoculated with RSV, with IAV, or with both viruses (RSV+IAV and IAV+RSV) administered sequentially, 24 h apart. On days 3 and 7 post-infection, lung tissues were processed for viral loads and immune cell populations. Lung functions were also evaluated. Mortality was observed only in the IAV+RSV group (50% of mice did not survive beyond 7 days). On day 3, the viral loads in single-infected and co-infected mice were not significantly different. However, on day 7, the IAV titer was much higher in the IAV+RSV group, and the RSV viral load was reduced. CD4 T cells were reduced in all groups on day 7 except in single-infected mice. CD8 T cells were higher in all experimental groups except the RSV-alone group. Increased airway resistance and reduced thoracic compliance were demonstrated in both co-infected groups. This model indicates that, among all the infection types we studied, infection with IAV followed by RSV is associated with the highest IAV viral loads and the most morbidity and mortality.

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Assessing Uncertainty in A2 Respiratory Syncytial Virus Viral Dynamics

Computational and Mathematical Methods in Medicine ◽

10.1155/2015/567589 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 15

Author(s):

Gilberto González-Parra ◽

Hana M. Dobrovolny

Keyword(s):

Respiratory Syncytial Virus ◽

Human Subjects ◽

Respiratory Illness ◽

The United States ◽

Viral Kinetics ◽

Phase Duration ◽

Syncytial Virus ◽

Parameter Values

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in children younger than 1 year of age in the United States. Moreover, RSV is being recognized more often as a significant cause of respiratory illness in older adults. Although RSV has been studied both clinically and in vitro, a quantitative understanding of the infection dynamics is still lacking. In this paper, we study the effect of uncertainty in the main parameters of a viral kinetics model of RSV. We first characterize the RSV replication cycle and extract parameter values by fitting the mathematical model to in vivo data from eight human subjects. We then use Monte Carlo numerical simulations to determine how uncertainty in the parameter values will affect model predictions. We find that uncertainty in the infection rate, eclipse phase duration, and infectious lifespan most affect the predicted dynamics of RSV. This study provides the first estimate of in vivo RSV infection parameters, helping to quantify RSV dynamics. Our assessment of the effect of uncertainty will help guide future experimental design to obtain more precise parameter values.

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