Standardized Phylogenetic Classification of Human Respiratory Syncytial Virus below the Subgroup Level.

A globally implemented unified phylogenetic classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. We formulated global consensus of HRSV classification on the basis of the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality curated dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to GenBank and GISAID (https://www.gisaid.org) public sequence databases through March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B and provided guidelines for defining prospective lineages. Our classification demonstrated robustness in its applicability to both complete and partial genomes. We envision that this unified HRSV classification proposal will strengthen HRSV molecular epidemiology on a global scale.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Severe Human Metapneumovirus and Group A Streptococcus Pneumonia in an Immunocompetent Adult.

An immunocompetent adult with asthma developed severe human metapneumovirus (HMPV) illness complicated by group A Streptococcus coinfection, progressing to acute respiratory distress syndrome and shock. Several coworkers had less severe HMPV infection. HMPV can cause severe respiratory illness in healthy adults and should be considered as a potential cause of community respiratory outbreaks.

Severe Respiratory Illness Associated with Human Metapneumovirus in Nursing Home, New Mexico, USA.

Human metapneumovirus is an emerging pathogen that causes upper and lower respiratory illness. Nursing home outbreaks of infection with this virus can cause severe illness and lead to poor patient outcomes. We report an outbreak investigation in a nursing home during 2018 and infection control guidelines to assist in disease control.

Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection.

BACKGROUND: Viral upper respiratory tract infections are associated with increased colonization by Streptococcus pneumoniae but the mechanisms underlying this relationship are unclear. The objective of this study is to describe a comprehensive picture of the cellular interaction between the adhering bacteria and host cells in the presence or absence of a viral co-infection. RESULTS: Gene expression profiles of Detroit-562 pharyngeal cells, which were either mock-infected or infected with human respiratory syncytial virus (RSV) or human parainfluenza virus 3 (HPIV3), were analyzed using human microarrays. Transcription response of S. pneumoniae strain TIGR4 (serotype 4) in the presence of either mock- or viral-infected cells was analyzed by pneumococcal microarray. Significantly regulated genes were identified by both significance analysis of microarray (SAM) and a ≥ 2-fold change ratio cut-off. The adherence of S. pneumoniae to human pharyngeal cells was significantly augmented in the presence of RSV or HPIV3 infection. Global gene expression profiling of the host cells during infection with RSV or HPIV3 revealed increased transcription of carcinoembryonic antigen-related cell adhesion molecules (CEACAM1), CD47, fibronectin, interferon-stimulated genes and many other host cell adhesion molecules. Pneumococci increased transcription of several genes involved in adhesive functions (psaA, pilus islet), choline uptake and incorporation (lic operon), as well as transport and binding. CONCLUSIONS: We have identified a core transcriptome that represents the basic machinery required for adherence of pneumococci to D562 cells infected or not infected with a virus. These bacterial genes and cell adhesion molecules can potentially be used to control pneumococcal adherence occurring secondary to a viral infection.

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

Respiratory Syncytial Virus Infection Among Hospitalized Infants in Four Middle-Income Countries.

BACKGROUND: Understanding respiratory syncytial virus (RSV) global epidemiology is important to inform future prevention strategies. METHODS: Hospitalized infants <1-year-old with acute illness were enrolled prospectively in Albania, Jordan, Nicaragua, and Philippines during respiratory seasons in 2015-2017. Medical chart review, parental interview, and post-discharge follow up were conducted. Respiratory specimens were tested using real-time RT-PCR for RSV. Infant characteristics associated with very severe illness (intensive care unit [ICU] admission or receipt of supplemental oxygen) were assessed using logistic regression to adjust for potential confounders (age, sex, study site, and preterm birth). RESULTS: Of 3634 enrolled hospitalized infants, 1129 (31%) tested positive for RSV. The median age of RSV-positive infants was 2.7 (IQR: 1.4-6.1) months and 665 (59%) were male. Very severe illness in 583 (52%) RSV-positive infants was associated with younger age (aOR 4.1, 95% CI: 2.6-6.5 for 0-2 compared to 9-11-months; P < .01), low weight-for-age z-score (aOR 1.9, 95% CI: 1.2-2.8; P < .01), ICU care after birth (aOR 1.6, 95% CI: 1.0-2.5; P = .048), and cesarean delivery (aOR 1.4, 95% CI: 1.0-1.8; P = .03). RSV subgroups A and B co-circulated at all sites with alternating predominance by year; subgroup was not associated with severity (aOR 1.0, 95% CI: 0.8-1.4). Nine (0.8%) RSV-positive infants died during admission or within ≤30 days of discharge, of which 7 (78%) were <6-months-old. CONCLUSIONS: RSV was associated with nearly a third of infant acute illness hospitalizations in four middle-income countries during the respiratory season, where, in addition to young age, factors including low weight-for-age might be important predictors of severity. RSV prevention strategies targeting young infants could substantially reduce RSV-associated hospitalizations in middle-income countries.

Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes.

Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with Ct values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with Ct values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a "flip-flop" phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Multiple Respiratory Syncytial Virus Introductions Into a Neonatal Intensive Care Unit.

BACKGROUND: Outbreaks of respiratory syncytial virus (RSV) in neonatal intensive care units (NICUs) are of concern because of the risk of severe disease in young infants. We describe an outbreak of RSV in a NICU and use whole genome sequencing (WGS) to better understand the relatedness of viruses among patients. METHODS: An investigation was conducted to identify patients and describe their clinical course. Infection control measures were implemented to prevent further spread. Respiratory specimens from outbreak-related patients and the community were tested using WGS. Phylogenetic trees were constructed to understand relatedness of the viruses. RESULTS: Seven patients developed respiratory symptoms within an 11-day span in December 2017 and were diagnosed with RSV; 6 patients (86%) were preterm and 1 had chronic lung disease. Three patients required additional respiratory support after symptom onset, and none died. Six of 7 patients were part of the same cluster based on > 99.99% nucleotide agreement with each other and 3 unique single-nucleotide polymorphisms were identified in viruses sequenced from those patients. The seventh patient was admitted from the community with respiratory symptoms and had a genetically distinct virus that was not related to the other 6. Implementation of enhanced infection control measures likely limited the spread. CONCLUSIONS: Using WGS, we found 2 distinct introductions of RSV into a NICU, highlighting the risk of healthcare-associated infections during RSV season. Early recognition and infection control measures likely limited spread, emphasizing the importance of considering RSV in the differential diagnosis of respiratory infections in healthcare settings.

Control of an outbreak of human parainfluenza virus 3 in hematopoietic stem cell transplant recipients.

Human parainfluenza virus 3 (HPIV3) infection can cause significant morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). There are no standard guidelines for the prevention and control of HPIV3 in the outpatient setting. After 2 HSCT inpatients diagnosed with HPIV3 were noted to have had multiple recent HSCT outpatient clinic (OPC) visits, an investigation of policy and procedures in the HSCT OPC was undertaken, and active surveillance for respiratory viral illness was instituted in the at-risk HSCT population. Between July 19 and August 30, 2005, 13 patients were diagnosed with HPIV3 infection. Morbidity in affected patients was significant, and mortality was high (38.5%) and not affected by antiviral therapy. Molecular typing identified several genetically distinct groups of the hemagglutinin-neuraminidase gene of the 11 available isolates. Based on sequence relatedness among the isolates and the demographic and exposure history of the patients, in many of these cases HPIV3 infection likely was acquired in the HSCT OPC. The major infection control interventions were introduced between August 20 and August 24. An epidemic curve revealed that HPIV3 infection frequency peaked between August 17 and August 26, with no cases identified after August 30. Prompt attention and focus on infection control interventions were associated with a rapid decrease in the number of incident cases. Policies and procedures regarding patients with respiratory viral illnesses in HSCT OPC populations should be formulated and universally reinforced with HSCT clinic staff to prevent the spread of these infections.

Results from the WHO external quality assessment for the respiratory syncytial virus pilot, 2016-17.

BACKGROUND: External quality assessments (EQAs) for the molecular detection of respiratory syncytial virus (RSV) are necessary to ensure the provision of reliable and accurate results. One of the objectives of the pilot of the World Health Organization (WHO) Global RSV Surveillance, 2016-2017, was to evaluate and standardize RSV molecular tests used by participating countries. This paper describes the first WHO RSV EQA for the molecular detection of RSV. METHODS: The WHO implemented the pilot of Global RSV Surveillance based on the WHO Global Influenza Surveillance and Response System (GISRS) from 2016 to 2018 in 14 countries. To ensure standardization of tests, 13 participating laboratories were required to complete a 12 panel RSV EQA prepared and distributed by the Centers for Disease Control and Prevention (CDC), USA. The 14th laboratory joined the pilot late and participated in a separate EQA. Laboratories evaluated a RSV rRT-PCR assay developed by CDC and compared where applicable, other Laboratory Developed Tests (LDTs) or commercial assays already in use at their laboratories. RESULTS: Laboratories performed well using the CDC RSV rRT-PCR in comparison with LDTs and commercial assays. Using the CDC assay, 11 of 13 laboratories reported correct results. Two laboratories each reported one false-positive finding. Of the laboratories using LDTs or commercial assays, results as assessed by Ct values were 100% correct for 1/5 (20%). With corrective actions, all laboratories achieved satisfactory outputs. CONCLUSIONS: These findings indicate that reliable results can be expected from this pilot. Continued participation in EQAs for the molecular detection of RSV is recommended.

Duplex real-time RT-PCR assay for detection and subgroup-specific identification of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region of the nucleoprotein gene combined in a single duplex reaction using all genome sequence data currently available in GenBank. The assay was validated for analytical sensitivity, specificity, reproducibility, and clinical performance with a geographically diverse collection of viral isolates and respiratory specimens in direct comparison with an established pan-HRSV rRT-PCR reference test. The assay was sensitive, reproducibly detecting as few as 5-10 copies/reaction of target RNA. The assay was specific, showing no amplification with a panel of 16 other common respiratory pathogens or predicted by in silico primer/probe analysis. The duplex rRT-PCR assay based on the most current available genome sequence data permits rapid, sensitive and specific detection and subgroup identification of HRSV.

Sequence analysis of the N, P, M and F genes of Canadian human metapneumovirus strains.

The complete nucleotide sequences of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) genes of 15 Canadian human metapneumovirus (hMPV) isolates were determined. Phylogenetic analysis revealed two distinct genetic clusters, or groups for each gene with additional sequence variability within the individual groups. Comparison of the deduced amino acid sequences for the N, M and F genes of the different isolates revealed that all three genes were well conserved with 94.1-97.6% identity between the two distinct clusters The P gene showed more diversity with 81.6-85.7% amino acid identity for isolates between the two clusters, and 94.6-100% for isolates within the same cluster.

Hospitalization due to human parainfluenza virus-associated lower respiratory tract illness in rural Thailand.

BACKGROUND: Human parainfluenza viruses (HPIVs) are an important cause of acute respiratory illness in young children but little is known about their epidemiology in the tropics. METHODS: From 2003-2007, we conducted surveillance for hospitalized respiratory illness in rural Thailand. We performed reverse-transcriptase polymerase chain reaction on nasopharyngeal specimens and enzyme immunoassay on paired sera. RESULTS: Of 10,097 patients enrolled, 573 (5%) of all ages and 370 (9%) of children <5 years of age had evidence of HPIV infection (HPIV1=189, HPIV2=54, HPIV3=305, untyped=27). Average adjusted annual incidence of HPIV-associated hospitalized respiratory illness was greatest in children aged <1 year (485 per 100,000 person years). CONCLUSIONS: In Thailand, HPIV caused substantial illnesses requiring hospitalization in young children.

Human bocavirus species 2 and 3 in Brazil.

BACKGROUND: The newly described human bocavirus (HBoV) species 2 and 3 have been repeatedly detected in stool strengthening the possibility that these viruses might present a tropism for the gastrointestinal tract and may be etiological agents of diarrhea. OBJECTIVE: In this study we assessed the presence of HBoV2 and HBoV3 in stool specimens from Brazilians with acute gastroenteritis. STUDY DESIGN: Stool samples from Brazilian patients with acute diarrhea were analyzed for HBoV2 and HBoV3 by PCR assay. Full or partial genome sequences were obtained for selected isolates. Electron microscopy analysis was used to investigate virus morphology. RESULTS: Electron microscopy confirmed the presence of virus-like particles in HBoV PCR-positive specimens, with morphology similar to other members of the Parvoviridae family. Five samples out of 807 (0.6%) were positive for HBoV3. Three of the HBoV3-positive patients were HIV/AIDS positive. A selected group of 144 samples was also tested for HBoV2 and 30 samples (20.8%) were positive, 11 of which were HIV/AIDS positive. CONCLUSION: This study reports the detection and genetic characterization of HBoV3 and HBoV2 in the stool of Brazilian patients with acute diarrhea. This is the first description of HBoV3 outside Australia, suggesting a wide global distribution of this virus. Further studies are needed to better understand the role of HBoV in gastrointestinal infections, particularly among patients with HIV/AIDS.

The burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural Thailand.

BACKGROUND: We describe the epidemiology of hospitalized RSV infections for all age groups from population-based surveillance in two rural provinces in Thailand. METHODS: From September 1, 2003 through December 31, 2007, we enrolled hospitalized patients with acute lower respiratory tract illness, who had a chest radiograph ordered by the physician, from all hospitals in SaKaeo and Nakhom Phanom Provinces. We tested nasopharyngeal specimens for RSV with reverse transcriptase polymerase chain reaction (RT-PCR) assays and paired-sera from a subset of patients with IgG enzyme immunoassay. Rates were adjusted for enrollment. RESULTS: Among 11,097 enrolled patients, 987 (8.9%) had RSV infection. Rates of hospitalized RSV infection overall (and radiographically-confirmed pneumonia) were highest among children aged<1 year: 1,067/100,000 (534/100,000 radiographically-confirmed pneumonia) and 1-4 year: 403/100,000 (222/100,000), but low among enrolled adults aged≥65 years: 42/100,000. Age<1 year (adjusted odds ratio [aOR]=13.2, 95% confidence interval [CI] 7.7, 22.5) and 1-4 year (aOR=8.3, 95% CI 5.0, 13.9) were independent predictors of hospitalized RSV infection. CONCLUSIONS: The incidence of hospitalized RSV lower respiratory tract illness among children<5 years was high in rural Thailand. Efforts to prevent RSV infection could substantially reduce the pneumonia burden in children aged<5 years.

Who gets hospitalized for influenza pneumonia in Thailand? Implications for vaccine policy.

Risk factor information for severe complications of interpandemic influenza is needed to inform vaccine policy in Thailand. We identified patients with lab-confirmed influenza who were hospitalized with pneumonia during September 2003 to August 2004. Among the 80 case-patients identified through a population-based pneumonia surveillance system in eastern Thailand, cases were 6.2 and 11.1 times more likely to be among persons<1 year old and >75 years old, respectively, compared with the overall population. Cases were also 7.6 times more likely to have chronic respiratory disease. In Thailand, the young, elderly, and those with chronic disease were at high risk for hospitalized pneumonia from influenza.

Detection of human metapneumovirus in clinical samples by immunofluorescence staining of shell vial centrifugation cultures prepared from three different cell lines.

Monoclonal antibody MAb-8 was evaluated for detection of human metapneumovirus (HMPV) in shell vial centrifugation cultures (SVCC). Detection of HMPV was similar in A549, HEp-2, and LLC-MK2 SVCC, and MAb-8 staining was optimal on day 2 postinoculation. Availability of SVCC for HMPV will be of significant benefit to clinical laboratories.

Sequence polymorphism of the predicted human metapneumovirus G glycoprotein.

The putative G glycoprotein genes of 25 human metapneumovirus (hMPV) field isolates obtained during five consecutive epidemic seasons (1997 to 2002) were sequenced. Sequence alignments identified two major genetic groups, designated groups 1 and 2, and two minor genetic clusters within each major group, designated subgroups A and B. Extensive nucleotide and deduced amino acid sequence variability was observed, consisting of high rates of nucleotide substitutions, use of alternative transcription-termination codons and insertions that retained the reading frame. Deduced amino acid sequences showed the greatest variability, with most differences located in the extracellular domain of the protein: nucleotide and amino acid sequence identities for the entire open reading frame ranged from 52 to 58 % and 31 to 35 %, respectively, between the two major groups. Like the closely related avian pneumovirus and human and bovine respiratory syncytial viruses, the predicted G protein of hMPV shared the basic features of a type II mucin-like glycosylated protein. However, differences from these related viruses were also observed, e.g. lack of conserved cysteine clusters as seen in human respiratory syncytial virus and avian pneumovirus. The displacement of genetic groups of hMPV observed during the study period suggests that potential antigenic differences in the G glycoprotein, which have evolved in response to immune-mediated pressure, may influence the circulation patterns of hMPV strains.