Valeria Falcone

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

ABSTRACT Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

Novel Human Metapneumovirus Sublineage

In a pediatric surveillance network, 287 (5.1%) of 5,580 specimens from patients with acute respiratory infections tested positive for human metapneumovirus (HMPV). Phylogenetic analysis of N- and F-gene sequences of identified HMPV showed that 30% belonged to a novel phylogenetic cluster.

Disseminated bocavirus infection after stem cell transplant.

To the Editor: Human bocavirus (HBoV) (1) is increasingly recognized as a cause of respiratory infections worldwide. Children and infants appear to be most at risk (2–7), although HBoV’s role in immunocompromised patients remains unclear. We report on a child with disseminated HBoV infection after hematopoietic stem cell transplantation (HSCT). HBoV DNA was detected at high levels in nasopharyngeal aspirates (NPAs) and in blood and stool samples. n nA 4.5-year-old boy with dyskeratosis congenita was brought for treatment to our hospital due to severe persistent cytopenia. Allogenic HSCT was performed in August 2006 after conditioning with total body irradiation (200 cGy, day –8 before HSCT surgery), fludarabine (days –7 to –4), antithymocyte globulin (days –4 to –1), and cyclophosphamide (days –3 to –2). He received 7.16 × 108 nucleated bone marrow cells/kg body weight from a 9/10 human leukocyte antigen–matched unrelated donor. Graft-versus-host disease (GvHD) prophylaxis consisted of a short course of methotrexate and cyclosporin A. Neutrophil and platelet engraftment occurred on days 22 and 65 after surgery, respectively. Despite pre- and post-HSCT anti-infective prophylaxis with cotrimoxazole, colistin, acyclovir, and fluconazole, Enterobacter cloacae sepsis was diagnosed on day 2. After meropenem treatment, blood cultures remained negative. On day 12, fever reoccurred, elevated C-reactive protein values (229 mg/L) and reduced general health were noted, but no bacterial pathogen was isolated. During this period, the patient received antimicrobial drug therapy with meropenem, tobramycin, vancomycin, and amphotericin B. On day 16, his body temperature peaked to 40.6°C, and a cough and dyspnea without wheezing developed. Chest radiograph results suggested pneumonia with perihilar infiltrates. Reduced oxygen saturation (pO2 86%) was recorded transcutaneously, and oxygen supplementation (maximum 4 L/min) was started by face mask (Appendix Figure). An NPA sample investigated by multiplex PCR (results provided by W. Puppe and J. Weigl; www.pid-ari.net) was negative for adenovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses 1–4, influenza viruses A and B, coronavirus, reovirus, enterovirus, Clamydia pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, B. parapertussis, and Legionella pneumophila, but positive for rhinovirus RNA. Retrospectively, the same NPA sample was reanalyzed for HBoV DNA by real-time PCR (7) and showed a viral load of 4.6 × 107 copies/mL (Appendix Figure); specificity was confirmed by sequencing. n nFrom day 19 on, the patient’s general health improved and the chest radiograph results returned to normal. After neutrophil engraftment (day 22) and addition of erythromycin to the antimicrobial drug regimen, body temperature decreased and oxygen supplementation was discontinued. However, rhinitis, cough, and low-grade fever (<38.5°C) persisted until day 50 (Appendix Figure), and HBoV DNA was detected in NPAs on days 37 and 44 at 2.4 × 1011 and 1.3 × 1014 copies/mL, respectively (Appendix Figure). The NPA sample on day 37 was still rhinovirus positive. n nConcurrent with the increased HBoV load in NPAs, cytomegalovirus (CMV) reactivation was first diagnosed by PCR on day 20 and peaked (58.250 copies/mL whole blood) on day 41 despite gancyclovir therapy. Switching to foscarnet led to temporary control of CMV replication (Appendix Figure). Additionally, on day 22, acute GvHD grade I with skin manifestations developed. Treatment with steroids until day 60 led to complete resolution. n nHBoV infection in this patient was not restricted to the respiratory tract. Diarrheic stool samples obtained on day 21 and, after resolution of respiratory symptoms, on day 75 showed substantial HBoV DNA (2.5 × 106 and 6.0 × 105 copies/mg, respectively; Appendix Figure). Tests for rotavirus and adenovirus antigens were negative, and no bacterial pathogen was isolated. Moreover, HBoV DNA was detected at lower levels (3.7 × 103 to 7.8 × 104 copies/mL) in 4 EDTA plasma samples taken days 21–47. Subsequent plasma (days 61, 68, 75, 88, 219), NPA (day 219), and stool (day 219) samples were negative for HBoV DNA. However, the ability of HBoV to cause persistent infection, as do other members of the Parvovirinae subfamily, cannot be excluded. Future investigations are needed to address this hypothesis. n nHere, we report on disseminated HBoV infection in an immunocompromised patient. Whether the clinical course in this case was more severe or prolonged than it would have been for HBoV infections in non-HSCT children remains unknown due to the lack of long-term observations in immunocompetent children. The dramatic increase of HBoV load in NPAs and viral dissemination most likely resulted from progressive impairment of cellular immunity as indicated by simultaneous CMV reactivation. Moreover, the increased viral load might have also been a consequence of steroid addition to immunosuppressive therapy to control GvHD. The contribution of HBoV to respiratory disease remains ambiguous because 2 NPA samples were also rhinovirus positive. Additional studies are required to investigate the pathogenic role of HBoV in double or multiple infections. Association of HBoV with the patient’s continued diarrhea is in accordance with previous studies (8–10). Prolonged fecal shedding has important implications for isolation measures in transplantation units. More studies in immunocompromised patients are required to evaluate the spectrum of pathology caused by this emerging virus.

Replication of primate foamy viruses in natural and experimental hosts.

Foamy viruses (FVs) are common apathogenic retroviruses readily spread by horizontal transmission in nonhuman primate and some other mammalian host populations. Primate FV infections have been known for half a century, i.e., 15 years before the definition of retroviruses and another 15 years before the detection of primate immune deficiency viruses. The emerging interest in human retroviruses included primate FV, and although the role of human hosts for FV was greatly overestimated temporarily, enthusiastic researchers compiled invaluable data on molecular biology and classic as well as molecular epidemiology of these viruses. It has been shown that lytic FV infection in a wide range of cell cultures is in great contrast to the silent state of the infection in animals. Once transmitted by saliva via biting, FVs reside in all tissues as DNA copies, but their replication is untraceable except in oral submucosal cells, which are thought to supply the virus for transmission. FVs have not definitely been associated with any disease, regardless of viral phylogenetic differences. Various primate and nonprimate species have been used for studies on the natural carrier state and primary infection. Experimental infections have mostly proven to be inefficient in primates as well as lower laboratory animals. However, investigation of the immune response in FV-infected animals has only partly explained the control of FV replication in the animal host. Thus, the biological role of FV remains an enigma to be resolved in the future.

Human metapneumovirus induces more severe disease and stronger innate immune response in BALB/c mice as compared with respiratory syncytial virus

BackgroundHuman metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are members of the Pneumovirinae subfamily of Paramyxoviridae and can cause severe respiratory disease, especially in infants and young children. Some differences in the clinical course of these infections have been described, but there are few comparative data on pathogenesis in humans and animal models. In this study, HMPV and RSV were compared for replication, pathogenesis and immune induction in BALB/c mice infected with equivalent inocula of either virus.MethodsViral titers in the lungs and in the nasal turbinates of mice were determined by plaque assay. Histopathological changes in the lungs as well as weight loss and levels of airway obstruction were monitored in the infected mice to record the severity of illness. Inflammatory cells recruited to the lungs were characterized by flow cytometry and by differential staining. In the case of natural killer cells, cytotoxic activity was also measured. Cytokine levels in the BAL were determined by cytometric bead array.ResultsRSV replicated to higher titers than HMPV in the lung and in the upper respiratory tract (URT), and virus elimination from the lungs was more rapid in HMPV-infected mice. Clinical illness as determined by airway obstruction, weight loss, and histopathology was significantly more severe after HMPV infection. A comparison of the cellular immune response revealed similar recruitment of T lymphocytes with a predominance of IFN-γ-producing CD8+ T cells. By contrast, there were obvious differences in the innate immune response. After HMPV infection, more neutrophils could be detected in the airways and there were more activated NK cells than in RSV-infected mice. This correlated with higher levels of IL-6, TNF-α and MCP-1.ConclusionThis study shows important differences in HMPV and RSV pathogenesis and suggests that the pronounced innate immune response observed after HMPV infection might be instrumental in the severe pathology.

Sequencing and Analysis of Globally Obtained Human Respiratory Syncytial Virus A and B Genomes

Background Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. Methods We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference. Results We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10-4 substitutions/site/year (95% HPD 5.61 × 10-4 to 7.6 × 10-4) and for RSVB the evolutionary rate was 7.69 × 10-4 substitutions/site/year (95% HPD 6.81 × 10-4 to 8.62 × 10-4). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB. Conclusions Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.

Human bocaviruses: Possible etiologic role in respiratory infection

Four species of human bocaviruses (HBoV) are currently included in the Bocavirus genus. There is satisfactory evidence demonstrating an association between HBoV1 and respiratory disease in children, and there is evidence that HBoV2 (and possibly the HBoV3 and HBoV4 species) are associated with gastroenteritis. In particular, HBoV1 has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. Virus persistence does play a role in the high frequency of co-infections with proper pathogens of the upper and lower respiratory tracts. The high detection rate of multiple respiratory viruses in up to 83% of respiratory specimens and the presence of asymptomatic HBoV1 infections complicate the elucidation of the pathogenic role of the agent. Overall, a large amount of data are available concerning HBoV1, whereas little information is available about other bocavirus species. High viral loads are often associated with symptoms, and viremia may be associated with systemic manifestations such as encephalopathy. The effects and mechanisms of latency, persistence, reactivation, and reinfection are poorly understood. Thus, particularly in co-infections, the pathogenic contribution of the detected bocavirus species cannot be accurately stated. This review summarizes the current knowledge of HBoV species and provides perspectives for future clinical studies.

Persistence of human bocavirus DNA in immunocompromised children.

Human bocavirus is frequently detected in immunocompetent as well as in immunocompromised children. However, the course of infection in immunocompromised children is still poorly investigated. In the present study, we describe 4 cases of repeat human bocavirus detection in the presence of severe immunodeficiency. In the view of homologous viral sequences identified in serial samples, possible persistence and reactivation in these patients are discussed.

Human bocavirus DNA in paranasal sinus mucosa.

To the Editor: Human bocavirus (HBoV) is a newly described parvovirus for which pathogenic potential has not clearly been elucidated (1). Recent findings suggest that HBoV may establish persistent infection of mucosal lymphocytes or contribute to tonsillar hyperplasia in children (2). In previous reports, we described prolonged HBoV DNA detection in immunocompromised children (3,4). Partial sequencing of the VP1 gene of HBoV from bronchoalveolar lavage fluid, plasma, and sphenoid sinus samples showed 100% identity, which suggested persistence of the same HBoV strain over a 5-month period (3). It remains speculative, however, whether paranasal sinus mucosa represents a site of HBoV persistence. To clarify this, we analyzed samples of paranasal mucosal tissue and nasal polyps from patients with chronic sinusitis for respiratory viruses and atypical bacteria. n nA total of 102 tissue samples were obtained from 88 patients (median age 48.5 years, range 13.3–88.1 years) from July 2009 through September 2010 after elective surgery. Indication for surgery was established by otorhinolaryngologists. The most common indication was chronic sinusitis. No patients displayed acute respiratory symptoms at the time of surgery. To detect asymptomatic shedding in the upper respiratory tract and viremia, we collected nasal swabs and EDTA-blood samples concurrently. The study protocol was approved by the Ethics Committee of the University of Freiburg. Informed written consent was obtained from all study participants. n nApproximately 25 mg of each tissue specimen was used for nucleic acid extraction by using an RNeasy Mini Kit, as described (5) (QIAGEN, Hamburg, Germany). To provide evidence that the QIAGEN RNeasy kit is also suitable for DNA extraction, we spiked HBoV negative samples with different amounts of HBoV DNA before extraction of nucleic acids was done with either the QIAGEN RNeasy Kit or DNA Blood Kit (QIAGEN). Extracted nucleic acids were then subjected to real-time PCR by using primers specific for HBoV. Minimal differences (±1 cycle threshold [Ct] value) in the HBoV PCR were detected; the QIAGEN RNeasy Kit was therefore used throughout the study (data not shown). Nasal swabs and ETDA-blood were purified by using a QIAamp MinElute Virus Spin Kit (QIAGEN). Multiplex PCR for respiratory viruses (Fast-track Diagnostics, Junglinster, Luxembourg) was conducted to detect influenza A (including pandemic [H1N1] 2009) and B viruses; respiratory syncytial virus; human metapneumovirus; HBoV; parainfluenza virus 1–4; human coronaviruses HKU1, NL63, 229E, and OC43; human rhinoviruses; human enteroviruses and parechoviruses; and adenoviruses. Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and Bordetella pertussis were analyzed as described (6–8). n nA single virus was detected in 22/102 (21.5%) tissue specimens, with HBoV being the most frequent (18/102, 17.6%), followed by rhinovirus (2/102, 1.9%), coronavirus 229E, and influenza A pandemic (H1N1) 2009 virus (1/102, 0.9%). HBoV was detected in specimens collected during July–September (13/18) and during February and March (5/18). All positive results were confirmed by single real-time PCR (9). For 14 patients, 2 different mucosal samples were tested and gave identical results. No multiple viral infections and no bacteria were detected. Median patient age was 51.2 years (range 14.4–74.2 years) for HBoV-positive and 47.6 years (range 13.3–88.1 years) for HBoV-negative samples. Ct analysis in single real-time PCR revealed a median Ct of 31 (range 28–38), corresponding to 200 genome equivalents/106 cells (range 3–1.8 × 104 copies/106 cells). No correlation between Ct value and patients’ age was observed (r2xa0=xa00.008; data not shown). No underlying disease was diagnosed for 13/18 HBoV-positive patients, whereas 2/18 and 3/18 patients had chronic obstructive pulmonary and oncologic disease, respectively. n nNasal swabs and EDTA-blood samples were obtained from 17/18 and 7/18 HBoV-positive patients, respectively. No HBoV was detected in any swabs or EDTA-blood samples available, indicating no virus shedding in the respiratory tract and no viremia. However, the 2 patients with rhinovirus-positive samples obtained from biopsy also had rhinovirus RNA detectable in nasal swabs, suggesting rhinovirus infection. Unfortunately, no nasal swab was available from the 2 patients whose sinus biopsy samples were positive for HCoV 229E and influenza A virus. n nIn this study, we simultaneously analyzed tissue specimens of paranasal sinuses and nasal polyps, as well as nasal swabs and blood samples, for a broad panel of viruses and atypical bacteria. To avoid seasonal bias, specimens were collected over a 1-year period and exclusively obtained from patients undergoing elective surgery in the absence of acute respiratory symptoms. n nThe finding that HBoV was present as a single virus in 18/22 virus-positive biopsy samples is intriguing. Moreover, the fact that no HBoV DNA was detected in nasal swabs or EDTA-blood samples indicates no active HBoV infection. In previous studies, HBoV DNA was frequently identified in the adenoids and tonsils of children (2,5,10). However, in contrast with our findings, detection of HBoV was mostly associated with other viruses, suggesting that co-virus–induced cellular damage might contribute to bocavirus reactivation and replication (5). Our findings indicate that persistence of viral nucleic acid in sinus mucosa might be a special advantage of HBoV, although the relevance of this observation remains unclear. Whether this presence as a single virus means a dead end for HBoV infection, true latency including the potential of reactivation, or a role in the pathogenesis of clinical conditions requiring surgery warrants future studies.

Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15

The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, nxa0=xa0563 patients) and the Xpert Flu/RSV XC assay (cohort 3, nxa0=xa0102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9xa0% (cohort 1), 95xa0% (cohort 2), and 98xa0% (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7xa0% and 100xa0% and specificities of 100xa0% and 94.1xa0%, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3xa0%. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary.