Persistent Airway Hyperresponsiveness Following Recovery from Infection with Pneumonia Virus of Mice.

Respiratory virus infections can have long-term effects on lung function that persist even after the acute responses have resolved. Numerous studies have linked severe early childhood infection with respiratory syncytial virus (RSV) to the development of wheezing and asthma, although the underlying mechanisms connecting these observations remain unclear. Here, we examine airway hyperresponsiveness (AHR) that develops in wild-type mice after recovery from symptomatic but sublethal infection with the natural rodent pathogen, pneumonia virus of mice (PVM). We found that BALB/c mice respond to a limited inoculum of PVM with significant but reversible weight loss accompanied by virus replication, acute inflammation, and neutrophil recruitment to the airways. At day 21 post-inoculation, virus was no longer detected in the airways and the acute inflammatory response had largely resolved. However, and in contrast to most earlier studies using the PVM infection model, all mice survived the initial infection and all went on to develop serum anti-PVM IgG antibodies. Furthermore, using both invasive plethysmography and precision-cut lung slices, we found that these mice exhibited significant airway hyperresponsiveness at day 21 post-inoculation that persisted through day 45. Taken together, our findings extend an important and versatile respiratory virus infection model that can now be used to explore the role of virions and virion clearance as well as virus-induced inflammatory mediators and their signaling pathways in the development and persistence of post-viral AHR and lung dysfunction.

Coinfection with Blood-Stage Plasmodium Promotes Systemic Type I Interferon Production during Pneumovirus Infection but Impairs Inflammation and Viral Control in the Lung.

Acute lower respiratory tract infections (ALRTI) are the leading cause of global childhood mortality, with human respiratory syncytial virus (hRSV) being a major cause of viral ALRTI in young children worldwide. In sub-Saharan Africa, many young children experience severe illnesses due to hRSV or Plasmodium infection. Although the incidence of malaria in this region has decreased in recent years, there remains a significant opportunity for coinfection. Recent data show that febrile young children infected with Plasmodium are often concurrently infected with respiratory viral pathogens but are less likely to suffer from pneumonia than are non-Plasmodium-infected children. Here, we hypothesized that blood-stage Plasmodium infection modulates pulmonary inflammatory responses to a viral pathogen but does not aid its control in the lung. To test this, we established a novel coinfection model in which mice were simultaneously infected with pneumovirus of mice (PVM) (to model hRSV) and blood-stage Plasmodium chabaudi chabaudi AS (PcAS) parasites. We found that PcAS infection was unaffected by coinfection with PVM. In contrast, PVM-associated weight loss, pulmonary cytokine responses, and immune cell recruitment to the airways were substantially reduced by coinfection with PcAS. Importantly, PcAS coinfection facilitated greater viral dissemination throughout the lung. Although Plasmodium coinfection induced low levels of systemic interleukin-10 (IL-10), this regulatory cytokine played no role in the modulation of lung inflammation or viral dissemination. Instead, we found that Plasmodium coinfection drove an early systemic beta interferon (IFN-ß) response. Therefore, we propose that blood-stage Plasmodium coinfection may exacerbate viral dissemination and impair inflammation in the lung by dysregulating type I IFN-dependent responses to respiratory viruses.

The effect of TIP on pneumovirus-induced pulmonary edema in mice.

BACKGROUND: Pulmonary edema plays a pivotal role in the pathophysiology of respiratory syncytial virus (RSV)-induced respiratory failure. In this study we determined whether treatment with TIP (AP301), a synthetic cyclic peptide that mimics the lectin-like domain of human TNF, decreases pulmonary edema in a mouse model of severe human RSV infection. TIP is currently undergoing clinical trials as a therapy for pulmonary permeability edema and has been shown to decrease pulmonary edema in different lung injury models. METHODS: C57BL/6 mice were infected with pneumonia virus of mice (PVM) and received TIP or saline (control group) by intratracheal instillation on day five (early administration) or day seven (late administration) after infection. In a separate set of experiments the effect of multiple dose administration of TIP versus saline was tested. Pulmonary edema was determined by the lung wet-to-dry (W/D) weight ratio and was assessed at different time-points after the administration of TIP. Secondary outcomes included clinical scores and lung cellular response. RESULTS: TIP did not have an effect on pulmonary edema in different dose regimens at different time points during PVM infection. In addition, TIP administration did not affect clinical severity scores or lung cellular response. CONCLUSION: In this murine model of severe RSV infection TIP did not affect pulmonary edema nor course of disease.

Cardiac dysfunction in pneumovirus-induced lung injury in mice.

OBJECTIVE: To determine biventricular cardiac function in pneumovirus-induced acute lung injury in spontaneously breathing mice. DESIGN: Experimental animal study. SETTING: Animal laboratory. SUBJECTS: C57Bl/6 mice. INTERVENTION: Mice were inoculated with the rodent pneumovirus, pneumonia virus of mice. MEASUREMENTS AND MAIN RESULTS: Pneumonia virus of mice-infected mice were studied for right and left ventricular function variables by high-field strength (7 Tesla) cardiac MRI at specific time points during the course of disease compared with baseline. One day before and at peak disease severity, pneumonia virus of mice-infected mice showed significant right and left ventricular systolic and diastolic volume changes, with a progressive decrease in stroke volume and ejection fraction. No evidence for viral myocarditis or viral presence in heart tissue was found. CONCLUSIONS: These findings show adverse pulmonary-cardiac interaction in pneumovirus-induced acute lung injury, unrelated to direct virus-mediated effects on the heart.

Toll-like receptor 7 gene deficiency and early-life Pneumovirus infection interact to predispose toward the development of asthma-like pathology in mice.

BACKGROUND: Respiratory tract viruses are a major environmental risk factor for both the inception and exacerbations of asthma. Genetic defects in Toll-like receptor (TLR) 7-mediated signaling, impaired type I interferon responses, or both have been reported in asthmatic patients, although their contribution to the onset and exacerbation of asthma remains poorly understood. OBJECTIVE: We sought to determine whether Pneumovirus infection in the absence of TLR7 predisposes to bronchiolitis and the inception of asthma. METHODS: Wild-type and TLR7-deficient (TLR7(-/-)) mice were inoculated with the rodent-specific pathogen pneumonia virus of mice at 1 (primary), 7 (secondary), and 13 (tertiary) weeks of age, and pathologic features of bronchiolitis or asthma were assessed. In some experiments infected mice were exposed to low-dose cockroach antigen. RESULTS: TLR7 deficiency increased viral load in the airway epithelium, which became sloughed and necrotic, and promoted an IFN-α/ß(low), IL-12p70(low), IL-1ß(high), IL-25(high), and IL-33(high) cytokine microenvironment that was associated with the recruitment of type 2 innate lymphoid cells/nuocytes and increased TH2-type cytokine production. Viral challenge of TLR7(-/-) mice induced all of the cardinal pathophysiologic features of asthma, including tissue eosinophilia, mast cell hyperplasia, IgE production, airway smooth muscle alterations, and airways hyperreactivity in a memory CD4(+) T cell-dependent manner. Importantly, infections with pneumonia virus of mice promoted allergic sensitization to inhaled cockroach antigen in the absence but not the presence of TLR7. CONCLUSION: TLR7 gene defects and Pneumovirus infection interact to establish an aberrant adaptive response that might underlie virus-induced asthma exacerbations in later life.

Pneumovirus in dogs with acute respiratory disease.

To determine which respiratory viruses circulate among confined dogs, we analyzed nasal and pharyngeal swab specimens from shelter dogs with acute respiratory disease. An unknown virus was isolated. Monoclonal antibody testing indicated that it was probably a pneumovirus. PCR and sequence analysis indicated that it was closely related to murine pneumovirus.

Granzyme A- and B-cluster deficiency delays acute lung injury in pneumovirus-infected mice.

Lower respiratory tract infection by the human pneumovirus respiratory syncytial virus is a frequent cause of acute lung injury in children. Severe pneumovirus disease in humans is associated with activation of the granzyme pathway by effector lymphocytes, which may promote pathology by exaggerating proapoptotic caspase activity and proinflammatory activity. The main goal of this study was to determine whether granzymes contribute to the development of acute lung injury in pneumovirus-infected mice. Granzyme-expressing mice and granzyme A- and B-cluster single- and double-knockout mice were inoculated with the rodent pneumovirus pneumonia virus of mice strain J3666, and were studied for markers of lung inflammation and injury. Expression of granzyme A and B is detected in effector lymphocytes in mouse lungs in response to pneumovirus infection. Mice deficient for granzyme A and the granzyme B cluster have unchanged virus titers in the lungs but show a significantly delayed clinical response to fatal pneumovirus infection, a feature that is associated with delayed neutrophil recruitment, diminished activation of caspase-3, and reduced lung permeability. We conclude that granzyme A- and B-cluster deficiency delays the acute progression of pneumovirus disease by reducing alveolar injury.

Animal models for studying respiratory syncytial virus infection and its long term effects on lung function.

BACKGROUND: Human respiratory syncytial virus (hRSV) infection causes a spectrum of illnesses ranging from mild infection to life-threatening bronchiolitis and respiratory failure. Human studies on the pathogenesis of RSV infection are invaluable, but animal models permit advances with the use of experimental strategies that would be inappropriate in human studies. METHODS: We review the advantages and disadvantages of various animal models for the study of hRSV infection. RESULTS: No animal model of hRSV infection replicates the complete spectrum of disease severity seen in humans. Available models differ in their ability to incorporate genetic technology and to allow the study of immunity, vaccine efficacy and treatment interventions. Although hRSV establishes disease in primates, this advantage is outweighed by the impracticalities and cost of using such models. The study of bovine RSV infection in calves is appealing because of parallels with human disease. Among rodent models, BALB/c mice have helped delineate the mechanisms underlying vaccine-enhanced RSV disease, and cotton rats have been used for preclinical testing. The single major disadvantage of studying hRSV in rodent models is the limited extent to which this host-restricted human pneumovirus replicates in mouse lung tissue. The rodent-specific Pneumovirus pathogen, pneumonia virus of mice, causes an infection that mirrors severe bronchiolitis and pneumonia in infants infected with RSV, including robust virus replication with profound inflammation. CONCLUSION: The recent development of the pneumonia virus of mice model has permitted exploration of the mechanisms of severe Pneumovirus disease in vivo with the use of sophisticated genetic tools and genetically manipulated mouse strains.

Pathogenicity, transmissibility, and tissue distribution of avian pneumovirus in turkey poults.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone. In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase-polymerase chain reaction and virus isolation. In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.

Reduced efficacy of hemorrhagic enteritis virus vaccine in turkeys exposed to avian pneumovirus.

Avian pneumovirus (APV) is an immunosuppressive respiratory pathogen of turkeys. We examined the effect of APV infection on the vaccine efficacy of hemorrhagic enteritis virus (HEV) vaccines. APV was inoculated in 2-wk-old turkeys. Two or four days later, an attenuated HEV vaccine (HEVp30) or marble spleen disease virus (MSDV) vaccine were administered. Virulent HEV challenge was given 19 days after HEV vaccination. APV exposure compromised the ability of HEVp30 and MSDV to protect turkeys against virulent HEV. The protective index values were as follows: MSDV (100%) versus APV + MSDV (0%) (P < 0.05); HEVp30 (60%) versus APV + HEVp30 (30%) (P < 0.05) (Experiment I) and HEVp30 (56%) versus APV + HEVp30 (20%) (P < 0.05) (Experiment II). These data indicated that APV reduced the efficacy of HEV vaccines in turkeys.

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli.

Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry. Experimental infections are typically less severe than those observed in field cases. Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents. Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV). In the current study, we examined the role of lentogenic NDV in the APV disease process. Two-week-old commercial turkey poults were infected with the Colorado strain of APV. Three days later, these poults received an additional inoculation of either NDV or E. coli. Dual infection of APV with either NDV or E. coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses. These lesions were not present in the single APV, NDV, or E coli groups. These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.

The clinical, pathological and microbiological outcome of an Escherichia coli O2:K1 infection in avian pneumovirus infected turkeys.

The purpose of this study was to evaluate the effect of an Escherichia coli infection in avian pneumovirus (APV)-infected turkeys. One group of 2-week-old specific pathogen-free (SPF) and two groups of 3-week-old conventional (CON) turkeys were inoculated oculonasally with virulent APV subtype A alone, with E. coli O2:K1 alone or with both agents at varying intervals (1, 3, 5 or 7 days) between the two inoculations. The birds were followed clinically and examined for macroscopic lesions at necropsy. Titres of APV were determined in the turbinates, trachea, lungs and air sacs. The number of E. coli O2:K1were assessed in the turbinates, trachea, lungs, air sacs, liver and heart. In both SPF and CON turkeys, dual infection resulted in an increased morbidity and a higher incidence of gross lesions compared to the groups given single infections, especially with a time interval between APV and E. coli inoculations of 3 and 5 days. APV was isolated from the respiratory tract of all APV-infected groups between 3 and 7 days post inoculation. E. coli O2:K1 was isolated only from turkeys that received a dual infection. It was recovered from the turbinates, trachea, lungs, heart and liver. These results show that APV may act as a primary agent predisposing to E. coli colonization and invasion.

Turkey rhinotracheitis virus and Escherichia coli experimental infection in chickens: histopathological, immunocytochemical and microbiological study.

The aim of this study was to evaluate the response of chickens to a combined infection with turkey rhinotracheitis virus (TRTV) and Escherichia coli O78:K80. Groups of specific-pathogen-free chickens were inoculated by eyedrop and intranasal routes with TRTV and/or E. coli O78:K80. Presence of E. coli O78:K80, histopathological changes and tissue distribution of viral antigen in the respiratory tract of chickens were evaluated. Dual infection resulted in increased severity of clinical signs, and macroscopic and microscopic lesions compared with those groups given single infections. All 36 chickens inoculated with TRTV plus E. coli O78:K80 showed severe rhinitis. Moreover, periorbital edema and fibrinous airsacculitis and pericarditis were observed in one of the three chickens inoculated with both agents and sacrificed at day 5 p.i. In addition, purulent material in the air spaces of the cranial bones was seen in three of the six animals from the same group sacrificed at days 5 and 7 p.i. The distribution of viral antigen in tissues was similar in groups inoculated with TRTV and TRTV plus E. coli, but viral antigen was detected only in main bronchi of chickens from the latter group. The quantity of E. coli O78:K80 isolated from the nasal cavity was greater in the group given dual infection. The results obtained suggest that TRTV may act as primary agent, enhancing E. coli multiplication. The lesions observed in the group inoculated with both agents could correspond to an initial stage of swollen head syndrome (SHS) and contribute to the hypothesis that SHS could be due to a mixed infection with TRTV and E. coli.

Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan.

Turkey rhinotracheitis (TRT) virus was first isolated from a commercial broiler chicken with swollen head syndrome (SHS) in Japan. At the same time, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian reovirus (ARV), Escherichia coli (E.coli), Morganella morganii, and Proteus mirabilis were also isolated from the same broiler chicken. The presence of antibodies to TRT virus was confirmed in the sera of 34-day-old chickens of the flock with SHS, however the antibodies to TRT virus were undetectable in the sera of 17-day-old chickens. In this investigation, we confirmed avian pneumovirus infection in chickens in Japan, and the virus and other agents may be considered as a cause of SHS.