Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus.

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.

Application of VirCapSeq-VERT and BacCapSeq in the diagnosis of presumed and definitive neuroinfectious diseases.

Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and the complexity of bioinformatic analysis. Here we report the use of virus capture-based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Thirty-four samples were categorized: (1) patients with definitive CNS infection by routine testing; (2) patients meeting clinically the Brighton criteria (BC) for meningoencephalitis; (3) patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT, and DNA extracts were used for BacCapSeq analysis. Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3, 11/26 samples (42%) were positive for at least one pathogen; however, 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess the clinical value of this novel technique, clinicians should understand the benefits and limitations of using this modality.

Compassionate-use pocapavir and immunoglobulin therapy for treatment of rituximab-associated enterovirus meningoencephalitis.

A 71-year-old woman previously on rituximab treatment for rheumatoid arthritis presented with 2 years of progressive neurologic symptoms. She was found to have persistent hypogammaglobulinemia and B cell depletion despite rituximab discontinuation a year prior. MRI revealed diffuse meningeal enhancement along the entire neuroaxis. LP showed a CSF lymphocytic pleocytosis, elevated protein, and presence of enterovirus by PCR. The patient was hospitalized several times for progressive clinical and radiologic decline, though she had transient improvements following treatment with immunoglobulin therapy. Her CSF remained positive for enterovirus PCR for at least 12 months. Though two brain biopsies were non-diagnostic, pan-Enterovirus was ultimately identified using a high-throughput next-generation sequencing technique. She was treated with compassionate-use pocapavir with clinical stabilization at 4-month follow-up; however, she expired 8 months later from a bacterial pneumonia.

SARS-CoV-2 Sequence Analysis during COVID-19 Case Surge, Liberia, 2021.

In June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases surged in Liberia. SARS-CoV-2 sequences from patients hospitalized during March-July 2021 revealed the Delta variant was in Liberia in early March and was dominant in June, irrespective of geography. Mutations and deletions suggest multiple SARS-CoV-2 Delta variant introductions.

The origins of G12P[6] rotavirus strains detected in Lebanon.

The G12 rotaviruses are an increasingly important cause of severe diarrhoea in infants and young children worldwide. Seven human G12P[6] rotavirus strains were detected in stool samples from children hospitalized with gastroenteritis in Lebanon during a 2011-2013 surveillance study. Complete genomes of these strains were sequenced using VirCapSeq-VERT, a capture-based high-throughput viral-sequencing method, and further characterized based on phylogenetic analyses with global RVA and vaccine strains. Based on the complete genomic analysis, all Lebanese G12 strains were found to have Wa-like genetic backbone G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Phylogenetically, these strains fell into two clusters where one of them might have emerged from Southeast Asian strains and the second one seems to have a mixed backbone between North American and Southeast Asian strains. Further analysis of these strains revealed high antigenic variability compared to available vaccine strains. To our knowledge, this is the first report on the complete genome-based characterization of G12P[6] emerging in Lebanon. Additional studies will provide important insights into the evolutionary dynamics of G12 rotaviruses spreading in Asia.

Association of Toll-like receptor 4 polymorphism with hepatitis E virus-infected Indian patients.

Hepatitis E infection caused by hepatitis E virus (HEV), a major public health concern in developing countries, is responsible for sporadic and epidemic acute viral hepatitis in adults. Pathogenesis of hepatitis E infection is poorly understood. Toll-like receptors (TLRs) are the key players of innate immunity recognize pathogen-associated molecular patterns (PAMPs). Previously, we found higher TLR4 expression (at protein and gene level) with impaired cytokine response upon stimulus of PBMCs with LPS in HEV-infected patients. In view of the earlier observations of the association of polymorphisms in TLR4 genes (A299G, C399T) with liver diseases, we investigated TLR4 polymorphisms in HEV-infected patients. We observed the significant association of TLR4-399CC and CT alleles with hepatitis E (both subclinical and acute patients). Carrier frequency of TLR4-399 CT was lower in patients' categories in comparison with the controls. Higher frequency of allele TLR4-399C significantly correlated with disease progression. Acute hepatitis E patients showed the higher frequency of CG and TA haplotypes, while the rare haplotype (TG) was more frequent in controls. The other single nucleotide polymorphism (SNP) at TLR4-299 (A>G) did not show any difference. We report here for the first time the association of TLR4 polymorphism with hepatitis E and suggest that TLR 4 hyporesponsiveness during HEV infection might be related to its polymorphism.

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy.

BACKGROUND: A 33 year-old pancreatic transplant recipient developed weakness, retinal blindness, and necrotic plaques on her face, scalp, and hands. METHODS: A muscle biopsy was analyzed by light and electron microscopy and high-throughput nucleic acid sequencing. RESULTS: The biopsy revealed microthrombosis and viral particles in swollen endothelial cell nuclei. High-throughput sequencing of nucleic acid revealed a novel polyomavirus. In situ hybridization confirmed the presence of the polyomavirus in endothelial cells at sites of myositis and cutaneous necrosis. CONCLUSIONS: New Jersey polyomavirus (NJPyV-2013) is a novel polyomavirus that may have tropism for vascular endothelial cells.

Multiplex PCR analysis of clusters of unexplained viral respiratory tract infection in Cambodia.

BACKGROUND: Fevers of unknown origin constitute a substantial disease burden in Southeast Asia. In majority of the cases, the cause of acute febrile illness is not identified. METHODS: We used MassTag PCR, a multiplex assay platform, to test for the presence of 15 viral respiratory agents from 85 patients with unexplained respiratory illness representing six disease clusters that occurred in Cambodia between 2009 and 2012. RESULTS: We detected a virus in 37 (44%) of the cases. Human rhinovirus, the virus detected most frequently, was found in both children and adults. The viruses most frequently detected in children and adults, respectively, were respiratory syncytial virus and enterovirus 68. Sequence analysis indicated that two distinct clades of enterovirus 68 were circulating during this time period. CONCLUSIONS: This is the first report of enterovirus 68 in Cambodia and contributes to the appreciation of this virus as an important respiratory pathogen.

Middle East respiratory syndrome coronavirus in bats, Saudi Arabia.

The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Molecular investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.

Serology-enabled discovery of genetically diverse hepaciviruses in a new host.

Genetic and biological characterization of new hepaciviruses infecting animals contributes to our understanding of the ultimate origins of hepatitis C virus (HCV) infection in humans and dramatically enhances our ability to study its pathogenesis using tractable animal models. Animal homologs of HCV include a recently discovered canine hepacivirus (CHV) and GB virus B (GBV-B), both viruses with largely undetermined natural host ranges. Here we used a versatile serology-based approach to determine the natural host of the only known nonprimate hepacivirus (NPHV), CHV, which is also the closest phylogenetic relative of HCV. Recombinant protein expressed from the helicase domain of CHV NS3 was used as antigen in the luciferase immunoprecipitation system (LIPS) assay to screen several nonprimate animal species. Thirty-six samples from 103 horses were immunoreactive, and viral genomic RNA was present in 8 of the 36 seropositive animals and none of the seronegative animals. Complete genome sequences of these 8 genetically diverse NPHVs showed 14% (range, 6.4% to 17.2%) nucleotide sequence divergence, with most changes occurring at synonymous sites. RNA secondary structure prediction of the 383-base 5' untranslated region of NPHV was refined and extended through mapping of polymorphic sites to unpaired regions or (semi)covariant pairings. Similar approaches were adopted to delineate extensive RNA secondary structures in the coding region of the genome, predicted to form 27 regularly spaced, thermodynamically stable stem-loops. Together, these findings suggest a promising new nonprimate animal model and provide a database that will aid creation of functional NPHV cDNA clones and other novel tools for hepacivirus studies.

Hepatitis E virus from India exhibits significant amino acid mutations in fulminant hepatic failure patients.

In India, hepatitis E virus (HEV) is the predominant cause of acute viral hepatitis (AVH) and fulminant hepatic failure (FHF) among pregnant women and adults. The present study evaluates association, if any, of the mutations in the viral genome with disease outcome. Ten genotype-1 complete genomes (five each from AVH and FHF patients) were sequenced. Phylogenetic analysis showed a distinct cluster including all five FHF-HEV sequences from western India (present study), one FHF isolate from northern India, and one AVH isolate detected in 2010 (present study). HEV genotype-1 sequences from fulminant cases exhibited 150 significantly different (p ≤ 0.05) nucleotide substitutions when compared to all genotype-1-AVH sequences as well as isolates from the Indian subcontinent. At six positions, all FHF sequences showed identical substitutions (1 non-synonymous). Six amino acid changes in ORF1; F179S, A317T, T735I, L1110F, V1120I, and F1439Y were significantly associated with HEV-type-1 FHF. The data suggests that the nucleotide substitutions recorded and/or L1110F and V1120I amino acid substitutions in helicase domain may play important role in determining outcome of HEV infection.

Application of VirCapSeq-VERT and BacCapSeq In the Diagnosis of Presumed and Definitive Neuroinfectious Diseases.

Background: Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and complexity of bioinformatic analysis. Here we report the use of virus capture based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Design/Methods: Thirty-four samples were categorized: (1) Patients with definitive CNS infection by routine testing; (2) Patients meeting clinically Brighton Criteria (BC) for meningoencephalitis (3) Patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT and DNA extracts were used for BacCapSeq analysis. Results: Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3,11/26 samples (42%) were positive for at least one pathogen, however 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. Conclusions: HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess clinical value of this novel technique, clinicians should understand benefits and limitations of using this modality.

High-throughput Sequencing to Identify Bacteremia in Children with Cancer.

Current methods to diagnose bacteremia are limited. In this pilot study of children with cancer presenting with fever, we determined the concordance between a novel high-throughput sequencing platform called BacCapSeq and blood culture. High-throughput sequencing had modest concordance with blood culture. Discordant organisms included those with both unlikely or potential clinical relevance.

Dynamics of SARS-CoV-2 variants characterized during different COVID-19 waves in Mali.

Background: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants may have contributed to prolonging the pandemic, and increasing morbidity and mortality related to coronavirus disease 2019 (COVID-19). This article describes the dynamics of circulating SARS-CoV-2 variants identified during the different COVID-19 waves in Mali between April and October 2021. Methods: The respiratory SARS-CoV-2 complete spike (S) gene from positive samples was sequenced. Generated sequences were aligned by Variant Reporter v3.0 using the Wuhan-1 strain as the reference. Mutations were noted using the GISAID and Nextclade platforms. Results: Of 16,797 nasopharyngeal swab samples tested, 6.0% (1008/16,797) tested positive for SARS-CoV-2 on quantitative reverse transcription polymerase chain reaction. Of these, 16.07% (162/1008) had a cycle threshold value ≤28 and were amplified and sequenced. The complete S gene sequence was recovered from 80 of 162 (49.8%) samples. Seven distinct variants were identified: Delta (62.5%), Alpha (1.2%), Beta (1.2%), Eta (30.0%), 20B (2.5%), 19B (1.2%) and 20A (1.2%). Conclusions and perspectives: Several SARS-CoV-2 variants were present during the COVID-19 waves in Mali between April and October 2021. The continued emergence of new variants highlights the need to strengthen local real-time sequencing capacity and genomic surveillance for better and coordinated national responses to SARS-CoV-2.

A potential role for SARS-CoV-2 small viral RNAs in targeting host microRNAs and modulating gene expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19) in humans, with symptoms ranging from mild to severe, including fatality. The molecular mechanisms surrounding the effects of viral infection on the host RNA machinery remain poorly characterized. We used a comparative transcriptomics approach to investigate the effects of SARS-CoV-2 infection on the host mRNA and sRNA expression machinery in a human lung epithelial cell line (Calu-3) and an African green monkey kidney cell line (Vero-E6). Upon infection, we observed global changes in host gene expression and differential expression of dozens of host miRNAs, many with known links to viral infection and immune response. Additionally, we discovered an expanded landscape of more than a hundred SARS-CoV-2-derived small viral RNAs (svRNAs) predicted to interact with differentially expressed host mRNAs and miRNAs. svRNAs are derived from distinct regions of the viral genome and sequence signatures suggest they are produced by a non-canonical biogenesis pathway. 52 of the 67 svRNAs identified in Calu-3 cells are predicted to interact with differentially expressed miRNAs, with many svRNAs having multiple targets. Accordingly, we speculate that these svRNAs may play a role in SARS-CoV-2 propagation by modulating post-transcriptional gene regulation, and that methods for antagonizing them may have therapeutic value.

Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses.

Enteroviruses are among the most common human viral pathogens. Infection with members of a subgroup of viruses within this genus, the nonpoliovirus enteroviruses (NPEVs), can result in a broad spectrum of serious illnesses, including acute flaccid myelitis (AFM), a polio-like childhood paralysis; neonatal sepsis; aseptic meningitis; myocarditis; and hand-foot-mouth disease. Despite the diverse primary sites of virus infection, including the respiratory and alimentary tracts, and an array of diseases associated with these infections, there is significant genetic and antigenic similarity among NPEVs. This conservation results in the induction of cross-reactive antibodies that are either able to bind and neutralize or bind but not neutralize multiple NPEVs. Using plaque reduction and enzyme-linked immunosorbent assay (ELISA)-based binding assays, we define the antigenic relationship among poliovirus and NPEVs, including multiple isolates of EV-D68, EV-A71, EV-D70, EV-94, EV-111, Coxsackievirus A24v, and rhinovirus. The results reveal extensive cross-reactivity among EVs that cannot be predicted from phylogenetic analysis. Determining the immunologic relationship among EVs is critical to understanding the humoral response elicited during homologous and heterologous virus infections. IMPORTANCE Enteroviruses (EVs) are common human pathogens. Although infection with EVs leads to cross-reactive antibodies, the clinical relevance of these antibodies is unclear given the estimated incidence of EV infections in the general population of one per year. The hypothesis that anti-EV cross-reactive antibodies can bind and neutralize heterologous EVs was investigated using polyclonal sera collected from animals immunized with individual EVs. Both binding and neutralization activities against heterologous EVs was observed in these sera, and we speculate that cross-reactive antibodies may modulate infection and disease severity. Defining the antigenic relationship among EVs may provide insights into the epidemiology and pathogenesis of enterovirus infections.

A Short Series of Case Reports of COVID-19 in Immunocompromised Patients.

Immunocompromised individuals are at risk of prolonged SARS-CoV-2 infection due to weaker immunity, co-morbidities, and lowered vaccine effectiveness, which may evolve highly mutated variants of SARS-CoV-2. Nonetheless, limited data are available on the immune responses elicited by SARS-CoV-2 infection, reinfections, and vaccinations with emerging variants in immunocompromised patients. We analyzed clinical samples that were opportunistically collected from eight immunocompromised individuals for mutations in SARS-CoV-2 genomes, neutralizing antibody (NAb) titers against different SARS-CoV-2 variants, and the identification of immunoreactive epitopes using a high-throughput coronavirus peptide array. The viral genome analysis revealed two SARS-CoV-2 variants (20A from a deceased patient and an Alpha variant from a recovered patient) with an eight amino-acid (aa) deletion within the N-terminal domain (NTD) of the surface glycoprotein. A higher NAb titer was present against the prototypic USA/WA1/2020 strain in vaccinated immunocompromised patients. NAb titer was absent against the Omicron variant and the cultured virus of the 20A variant with eight aa deletions in non-vaccinated patients. Our data suggest that fatal SARS-CoV-2 infections may occur in immunocompromised individuals even with high titers of NAb post-vaccination. Moreover, persistent SARS-CoV-2 infection may lead to the emergence of newer variants with additional mutations favoring the survival and fitness of the pathogen that include deletions in NAb binding sites in the SARS-CoV-2 surface glycoprotein.

Detection of antibodies to Anaplasma phagocytophilum and Babesia microti using linear peptides.

Anaplasma phagocytophilum and Babesia microti are emerging tick-borne pathogens in the United States. Although active infection is typically diagnosed by direct diagnostic tests, such as blood smear or polymerase chain reaction assay, serologic assays can be helpful to identify past infections, and the use of acute plus convalescent testing can potentially identify recent infections. We employed a peptide array to select sets of linear peptides for serologic diagnosis of infections with A. phagocytophilum and B. microti. Three optimal peptides were selected for each agent based on their performance with clinical specimens. All three A. phagocytophilum peptides were located within the conserved fragments of the MSP2 antigen. Two B. microti peptides were located in the N terminus of the SA-1 antigen; the third was in the BMN 1-17 antigen. We found that these peptides can be a useful tool for detection of antibody reactivity to both of these pathogens.

Human Polyomavirus 9-An Emerging Cutaneous and Pulmonary Pathogen in Solid Organ Transplant Recipients.

IMPORTANCE: We describe the first report to our knowledge of cutaneous and systemic pathogenicity of human polyomavirus 9 in solid organ transplant recipients. OBJECTIVE: Three solid organ transplant recipients developed a widespread, progressive, violaceous, and hyperkeratotic skin eruption. All died from pulmonary and multiorgan failure around 1 year from onset of the rash. Routine clinical diagnostic testing could not identify any causative agent; therefore, samples and autopsies were investigated for novel pathogens using high-throughput sequencing. DESIGN, SETTING, AND PARTICIPANTS: This case series, including 3 solid organ transplant recipients who developed characteristic pink, violaceous, or brown hyperkeratotic papules and plaques throughout the body, was conducted at the Columbia University Medical Center. Lesional skin biopsies were collected from all 3 patients and subjected to high-throughput illumina sequencing for identification of microbial pathogens. Human polyomavirus 9 was identified in lesional skin biopsies. We subsequently collected ocular swabs, oral swabs, urine samples, and blood samples from patients, and organ tissues at autopsy in 1 patient. We investigated these samples for the presence of human polyomavirus 9 using in situ hybridization and quantitative polymerase chain reaction (PCR) assays. MAIN OUTCOMES AND MEASURES: A description of the clinical and pathologic findings of 3 patients. RESULTS: This case series study found that human polyomavirus 9 was detected in the skin biopsies of all 3 patients by a capture-based high-throughput sequencing method platform (VirCapSeq-VERT). Human polyomavirus 9 was also detected in blood, oral, ocular swabs, and urine by real-time polymerase chain reaction (PCR) assay. In situ hybridization and quantitative PCR assays were performed on the skin biopsies from 3 patients and lung autopsy of 1 patient, which showed the presence of human polyomavirus 9 messenger RNA transcripts, indicating active viral replication and pathogenesis in the skin and lungs. CONCLUSIONS AND RELEVANCE: Human polyomavirus 9 was associated with the widespread cutaneous eruption. All 3 patients had progression of cutaneous disease, accompanied by clinical deterioration, pulmonary failure, and death. One patient underwent autopsy and human polyomavirus 9 was identified in the lungs and paratracheal soft tissue. These findings suggest that human polyomavirus 9 may be associated with cutaneous and possibly pulmonary infection and death in solid organ transplant recipients.