Respiratory Disease In Mice Research Articles

Direct monitoring of meropenem therapeutic efficacy against Klebsiella pneumoniae respiratory infection by bioluminescence imaging.

Introduction. Klebsiella pneumoniae is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection. K. pneumoniae causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates.Hypothesis. There is a growing concern about the emergence of multidrug-resistant K. pneumoniae strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents.Aim. K. pneumoniae causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent Klebsiella to monitor therapeutic efficacy.Methodology. We engineered a bioluminescence reporter strain of K. pneumoniae to monitor the impact of antibiotics in a murine respiratory disease model.Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo. Light production is directly linked to bacterial viability, and this novel bioluminescent K. pneumoniae strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung.Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.

Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice.

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.

Sir Henry Harris. 28 January 1925—31 October 2014

Henry Harris was the first member of his family to attend university, where he initially read modern languages before embarking on medicine. During the course of his medical studies he developed a passion for experimental work that led him to forsake clinical medicine for a career in research. His most significant and enduring legacy was the demonstration that a virus causing respiratory disease in mice could be used to fuse cells together almost regardless of type or species of origin. This not only opened up novel approaches to the study of both normal and cancer cell genetics and biology but also enabled the production of monoclonal antibodies, reagents that are of increasing value clinically as well as in biomedical research. From very early in his career he showed striking independence in the choice of topics that he regarded as important and interesting, entirely ignoring evanescent trends that determine the course of all too many research careers. Moreover, he continued to engage in benchwork up to, and even beyond, retirement.

Adaptive evolution influences the infectious dose of MERS-CoV necessary to achieve severe respiratory disease

We recently established a mouse model (288–330+/+) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 106 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 103 and 105 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10–1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease.

Virulence, pathology, and pathogenesis of Pteropine orthoreovirus (PRV) in BALB/c mice: Development of an animal infection model for PRV.

BackgroundCases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus Orthoreovirus (family: Reoviridae) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated.MethodsThe virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [Eonycteris spelaea] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods.ResultsThe intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used.ConclusionsThe BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch’s postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV.

Full-Genome Analysis of a Canine Pneumovirus Causing Acute Respiratory Disease in Dogs, Italy

An outbreak of canine infectious respiratory disease (CIRD) associated to canine pneumovirus (CnPnV) infection is reported. The outbreak occurred in a shelter of the Apulia region and involved 37 out of 350 dogs that displayed cough and/or nasal discharge with no evidence of fever. The full-genomic characterisation showed that the causative agent (strain Bari/100-12) was closely related to CnPnVs that have been recently isolated in the USA, as well as to murine pneumovirus, which is responsible for respiratory disease in mice. The present study represents a useful contribution to the knowledge of the pathogenic potential of CnPnV and its association with CIRD in dogs. Further studies will elucidate the pathogenicity and epidemiology of this novel pneumovirus, thus addressing the eventual need for specific vaccines.

Lung Cytokine Gene Expression is Correlated with Increased Severity of Disease in a Novel H4N8 Influenza Virus Isolated from Shorebirds

ABSTRACTThe lung cytokine gene expression profiles of mice infected with 2 strains of H4N8 viruses isolated from shorebirds and reference H4 viruses from ducks are compared. Major differences between the two H4N8 strains of shorebirds, one of which causes a severe respiratory disease in mice, are in the PB1 and NS1 genes. In mice with H4N8 virus induced pneumonia, overall expression of TNF-α, IL-6 and IL-12 genes was markedly higher than in mice infected with other H4 viruses tested, although gene expression of type I interferon was not increased until day 4 post viral infection. In contrast, in mice infected with a comparison H4N8 strain, gene expression of type I interferon peaked on day 1 post viral infection. Overall, the cytokine response corresponds with the severity of disease caused by shorebird H4N8 virus. The results obtained in this study provide valuable information to understand the immunopathology induced by a low pathogenic avian influenza virus, which may be useful in preparation for outbreaks of novel influenza A virus.

Evaluation of pneumonia virus of mice as a possible human pathogen.

Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.

H4N8 subtype avian influenza virus isolated from shorebirds contains a unique PB1 gene and causes severe respiratory disease in mice

H4N8 subtype avian influenza viruses were isolated from shorebirds in eastern Hokkaido. All the isolates shared >99.7% nucleotide homology, and all the viral genes except for PB1 were highly related to those of A/red-necked stint/Australia/1/04. Thus, the isolates were regarded as PB1 reassortants. The most similar PB1 gene was identified in A/mallard/New Zealand/1615-17/04 (H4N6) with nucleotide homology of 90.9%. BALB/c mice intranasally inoculated with the H4N8 isolates developed severe respiratory disease, which eventually led to death in some mice. The virus was isolated from the lungs, and viral antigen was detected in the lungs with pneumonia. Other H4 subtype viruses tested did not cause any symptoms in mice, although these viruses were also isolated from the lungs. The PB2 gene of the H4N8 isolates contains K482R, but not the E627K or D701N substitutions. The PB1-F2 gene of the isolates consists of a 101-amino acid unique sequence, but lacks the N66S mutation.

Critical requirement for 4‐1BBL for protection from severe but not mild respiratory Influenza A infection in mice

In view of intensifying concerns about pandemic Influenza virus, there is considerable interest in the mechanisms that control respiratory Influenza virus infection. Here we sought to determine the importance of 4‐1BBL‐mediated costimulation during mild versus severe respiratory Influenza virus infection in mice. During infection with the mildly virulent Influenza A/HKx31 virus, regardless of route of infection or epitope studied, 4‐1BBL is completely dispensable for the primary CD8 T cell response. However, 4‐1BBL is required for maximal recall responses, previously attributed to a requirement for 4‐1BBL in the maintenance of T cell memory. In contrast, during primary intranasal infection with Influenza A/PR8, which leads to severe respiratory disease in mice, the absence of 4‐1BBL results in decreased CD8 T cell responses in the lung, increased viral load and increased mortality. Thus 4‐1BBL plays a key role in protective immunity in the respiratory tract during severe Influenza disease. These results show that costimulation requirements are greater during severe as compared to mild respiratory Influenza virus infection.

Gender is a major factor in determining the severity of mycoplasma respiratory disease in mice.

Gender is a significant factor in determining the susceptibility to and severity of pulmonary diseases in both humans and animals. Murine respiratory mycoplasmosis (MRM), due to Mycoplasma pulmonis infection, is an excellent animal model for evaluation of the role of various host factors on the development of acute or chronic inflammatory lung diseases. MRM has many similarities to mycoplasma respiratory disease in humans. The purpose of the present study was to determine whether gender has a significant impact on lung disease due to M. pulmonis infection in mice. It was demonstrated that male mice consistently developed more severe disease in the lung parenchyma than did female mice. There was no gender difference in disease severity along the airways or any difference in mycoplasma numbers in lungs of male and female mice. Furthermore, surgical removal of reproductive organs reduced the severity of mycoplasma disease and the numbers of mycoplasma organisms recovered from lungs. Thus, gender plays a significant role in determining the severity of M. pulmonis disease. In fact, the gender of the host was a major factor in determining whether an acute or chronic inflammatory lung disease developed after infection with M. pulmonis.

Protective effect of vaccines on Mycoplasma pulmonis-induced respiratory disease of mice.

Mice inoculated intranasally with either a virulent or an avirulent strain of live Mycoplasma pulmonis were resistant to respiratory disease induced by a subsequent intranasal challenge with virulent organisms. Similarly, mice inoculated intravenously with the virulent strain were resistant to intranasal challenge with the same strain. In contrast, mice inoculated intravenously with avirulent M. pulmonis were not resistant to intranasal challenge with the virulent mycoplasma strain. Studies on mice inoculated intravenously with the two strains of M. pulmonis indicated that persistance of mycoplasmas in the respiratory tract may be important in inducing resistance to intranasal challenge with M. pulmonis. These observations, together with the lack of correlation between the level of serum antibodies and resistance to M. pulmonis-induced respiratory disease, suggested that local immune mechanisms were important in resistance. It is proposed that an effective vaccination schedule to protect mice against M. pulmonis-induced respiratory disease may be one that stimulates both systemic and local immune defenses. This suggestion is supported by the observation that systemic followed by local administration of inactivated M. pulmonis was more effective in inducing resistance in mice to intranasal challenge with live organisms than was systemic administration alone. In addition, mice inoculated solely by the intranasal route with inactivated mycoplasmas were resistant to M. pulmonis-induced respiratory disease. These studies indicate the importance of local defense mechanisms in the induction of resistance to M. pulmonis-induced respiratory disease in mice.