Eptesicus fuscus Orthorubulavirus, a Close Relative of Human Parainfluenza Virus 4, Discovered in a Bat in South Dakota.

Bats are a reservoir for many zoonotic viruses and host large numbers of genetically diverse species in the families Rhabdoviridae, Coronaviridae, and Paramyxoviridae. Viruses from these families have repeatedly spilled over to humans in recent decades, causing significant clinical disease and deaths. Here, metagenomic sequencing of a big brown bat (Eptesicus fuscus) submitted for rabies testing due to human exposure identified a novel paramyxovirus, Eptesicus fuscus orthorubulavirus (EfORV), in South Dakota, United States. The nearly complete 15,814-nucleotide genome shared 72% identity with that of human parainfluenza virus 4 (HPIV4), a virus that causes significant clinical disease, typically bronchiolitis and pneumonia, in children less than 2 years of age. Phylogenetic analysis confirmed a close evolutionary history between EfORV and HPIV4, reminiscent of other orthorubulaviruses with highly similar bat and mammalian species, including conspecific human and bat mumps virus, mammalian parainfluenza virus 5 and bat Alston virus, and porcine La Piedad Michoacán virus and bat Mapuera virus. These results support the idea that bats are a reservoir for diverse paramyxoviruses with closely shared evolutionary histories, compared with a number of significant human pathogens, and expand the range of bat paramyxoviruses to North America. Given the propensity of paramyxoviruses to overcome species barriers, additional surveillance and characterization of EfORV are warranted. IMPORTANCE Bats are a reservoir of large numbers of viruses. Among bat-borne zoonotic viruses, members of Coronaviridae and Paramyxoviridae have had the largest impact on human health. The repeated spillover of bat viruses to humans, often with devastating results, has led to increased surveillance and virus discovery efforts in hot spots for virus emergence, largely Asia and Africa. Apart from rabies virus, little surveillance of viruses in bats is performed in North America. Here, viral metagenomic sequencing identified a close relative to HPIV4 in a big brown bat found in a motel room in South Dakota. The virus, EfORV, was 72% identical to HPIV4, which causes clinically significant respiratory disease, mainly in children; it represents the first bat paramyxovirus identified in North America. Close genetic relationships between bat and mammalian orthorubulaviruses underscore the importance of bats as a reservoir for zoonotic viruses.

Genetic analysis of human parainfluenza virus type 4 associated with severe acute respiratory infection in children in Luohe City, Henan Province, China, during 2017-2018.

During 2017-2018, nasopharyngeal aspirates (NPAs) from 627 hospitalized patients with severe acute respiratory infection at Luohe Center Hospital were tested by RT-PCR for human parainfluenza virus 4 (HPIV-4). Fourteen (2.2%) of the 627 samples were positive for HPIV-4. The complete HN gene was amplified from nine positive samples and sequenced. Sequence comparisons showed that the HPIV-4 strains circulating in the city of Luohe are closely related to HPIV-4A strains. Our study indicated that there were multiple lineages of HPIV-4 circulating in Henan Province in China during the study period. This will improve our understanding of the epidemiological and clinical characteristics of HPIV-4.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Human parainfluenza virus surveillance in pediatric patients with lower respiratory tract infections: a special view of parainfluenza type 4 / Vigilância dos vírus parainfluenza humanos em pacientes pediátricos com infecções do trato respiratório inferior: uma visão especial do parainfluenza tipo 4

Abstract Objective: Characterize the role of human parainfluenza virus and its clinical features in Brazilian children under 2 years of age presenting with acute lower respiratory tract infections. Methods: Real-time assays were used to identify strains of human parainfluenza virus and other common respiratory viruses in nasopharyngeal aspirates. One thousand and two children presenting with acute lower respiratory tract illnesses were enrolled from February 2008 to August 2010. Results: One hundred and four (10.4%) patients were human parainfluenza virus positive, of whom 60 (57.7%) were positive for human parainfluenza virus-3, 30 (28.8%) for human parainfluenza virus-4, 12 (11.5%) for human parainfluenza virus-1, and two (1.9%) for human parainfluenza virus-2. Seven (6.7%) patients had more than one strain of human parainfluenza virus detected. The most frequent symptoms were tachypnea and cough, similar to other viral respiratory infections. Clinical manifestations did not differ significantly between human parainfluenza virus-1, -2, -3, and -4 infections. Human parainfluenza virus-1, -3, and -4 were present in the population studied throughout the three years of surveillance, with human parainfluenza virus-3 being the predominant type identified in the first two years. Conclusion: Human parainfluenza viruses contribute substantially to pediatric acute respiratory illness (ARI) in Brazil, with nearly 30% of this contribution attributable to human parainfluenza virus-4.

Resumo Objetivo: Caracterizar o papel do VPH-4 e suas características clínicas em crianças brasileiras com menos de dois anos de idade com infecções agudas do trato respiratório inferior. Métodos: Ensaios em tempo real foram utilizados para identificar tipos de VPH e outros vírus respiratórios comuns em aspirados nasofaríngeos. Mil e duas crianças com doença aguda do trato respiratório inferior foram inscritas para participar de fevereiro de 2008 a agosto de 2010. Resultados: 104 (10,4%) pacientes eram VPH positivos, dos quais 60 (57,7%) eram positivos para VPH-3, 30 (28,8%) para VPH-4, 12 (11,5%) para VPH-1 e dois (1,9%) para VPH-2. Sete (6,7%) pacientes apresentaram mais de um tipo de VPH detectado. Os sintomas mais frequentes foram tosse e taquipneia, semelhantes a outras infecções respiratórias virais. As manifestações clínicas não diferiram de forma significativa entre as infecções por VPH-1, -2, -3 e -4. Os VPH-1, -3 e -4 estavam presentes na população estudada ao longo dos três anos de vigilância, e o VPH-3 foi o tipo predominante identificado nos primeiros dois anos. Conclusão: Os VPHs contribuem substancialmente para a DRA pediátrica no Brasil com quase 30% dessa contribuição atribuível ao VPH-4.

Clinical and Molecular Epidemiology of Human Parainfluenza Viruses 1-4 in Children from Viet Nam.

HPIVs are serologically and genetically grouped into four species that account for up to 10% of all hospitalizations due to acute respiratory infection in children under the age of five. Genetic and epidemiological data for the four HPIVs derived from two pediatric cohorts in Viet Nam are presented. Respiratory samples were screened for HPIV1-4 by real-time PCR. Demographic and clinical data of patients infected with different HPIV were compared. We used a hemi-nested PCR approach to generate viral genome sequences from HPIV-positive samples and conducted a comprehensive phylogenetic analysis. In total, 170 samples tested positive for HPIV. HPIV3 was most commonly detected in our cohort and 80 co-detections of HPIV with other respiratory viruses were found. Phylogenetic analyses suggest local endemic circulation as well as punctuated introductions of new HPIV lineages. Viral gene flow analysis revealed that Viet Nam is a net importer of viral genetic diversity. Epidemiological analyses imply similar disease severity for all HPIV species. HPIV sequences from Viet Nam formed local clusters and were interspersed with sequences from diverse geographic regions. Combined, this new knowledge will help to investigate global HPIV circulation patterns in more detail and ultimately define more suitable vaccine strains.

Human parainfluenza virus surveillance in pediatric patients with lower respiratory tract infections: a special view of parainfluenza type 4.

OBJECTIVE: Characterize the role of human parainfluenza virus and its clinical features in Brazilian children under 2 years of age presenting with acute lower respiratory tract infections. METHODS: Real-time assays were used to identify strains of human parainfluenza virus and other common respiratory viruses in nasopharyngeal aspirates. One thousand and two children presenting with acute lower respiratory tract illnesses were enrolled from February 2008 to August 2010. RESULTS: One hundred and four (10.4%) patients were human parainfluenza virus positive, of whom 60 (57.7%) were positive for human parainfluenza virus-3, 30 (28.8%) for human parainfluenza virus-4, 12 (11.5%) for human parainfluenza virus-1, and two (1.9%) for human parainfluenza virus-2. Seven (6.7%) patients had more than one strain of human parainfluenza virus detected. The most frequent symptoms were tachypnea and cough, similar to other viral respiratory infections. Clinical manifestations did not differ significantly between human parainfluenza virus-1, -2, -3, and -4 infections. Human parainfluenza virus-1, -3, and -4 were present in the population studied throughout the three years of surveillance, with human parainfluenza virus-3 being the predominant type identified in the first two years. CONCLUSION: Human parainfluenza viruses contribute substantially to pediatric acute respiratory illness (ARI) in Brazil, with nearly 30% of this contribution attributable to human parainfluenza virus-4.

Human parainfluenza virus infection in severe acute respiratory infection cases in Beijing, 2014-2016: A molecular epidemiological study.

BACKGROUND: Severe acute respiratory infection (SARI) threatens human health and even survival, causing a huge number of hospitalized patients every year. However, as one of the most common respiratory viruses circulated worldwide, the epidemiological and phylogenetic characteristics of human parainfluenza virus (HPIV) in these cases were not well known. OBJECTIVES: To reveal the epidemiological features of HPIV infection in SARIs in Beijing area from September 2014 to August 2016. METHODS: A total of 1229 SARI cases in Beijing area were enrolled, investigated, sampled, and tested by multiplex real-time PCR to identify HPIVs and other common respiratory viruses. Eighteen HPIV-3 viruses isolated from all HPIV-positive samples in these SARI cases were sequenced and analyzed. RESULTS: Among all enrolled cases, 0.81%, 0.73%, 4.48%, and 0.57% were positive for HPIV-1 to HPIV-4, respectively. The highest yield rate of HPIV infection occurred in children under 5 years old (9.07%), followed by the patients over 60 years old (6.02%). The phylogenetic information of HPIV-3 showed that all viruses belonged to Cluster C3a. CONCLUSIONS: Besides the young children, the elders older than 60 years also showed a relatively high infection rate of HPIVs, which should be given comparable attentions. Moreover, the HPIV-3 circulating in China undergoes continued evolution, suggesting the potential risk of evolved HPIV infection should not be overlooked.

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

Establishment of reverse transcription polymerase chain reaction assay for simultaneous detection of human parainfluenza virus types 1 to 4 in children with influenza like illnesses.

OBJECTIVES: The aim of this study was to develop an in-house multiplex reverse transcription polymerase chain reaction (mRT-PCR), which can recognize HPIV1-4 in clinical samples. BACKGROUND: Human parainfluenza virus (HPIV) is one of the major causes of viral respiratory infections and can affect people at any age, especially infants and young children. METHODS: Four sets of specific primers targeting conserved areas of hemagglutinin-neuraminidase (HN) genes of HPIV1-4, were designed and tested with type-related plasmid controls. Specificity and sensitivity of mPCR were tested. One-step mRT-PCR was set up using a viral panel containing 10 respiratory viruses, including HPIVs. One hundred nasopharyngeal samples of respiratory infection patients were tested using the set One-step mRT-PCR. RESULTS: The specificity of set mPCR for HPIV1-4 using plasmid positive controls was proved and reaction sensitivity was measured. The specificity of set mRT-PCR was confirmed and 4 and 5 out of 100 clinical samples were HPIV1 and HPIV2 positive, respectively. CONCLUSION: The developed one-step mRT-PCR in this study is an effective and specific assay for clinical diagnosis of HPIV1 to 4 (Tab. 1, Fig. 6, Ref. 28).

Metagenomics Study of Viral Pathogens in Undiagnosed Respiratory Specimens and Identification of Human Enteroviruses at a Thailand Hospital.

Numerous pathogens cause respiratory infections with similar symptoms. Routine diagnostics detect only a limited number of pathogens, leaving a gap in respiratory illness etiology surveillance. This study evaluated next-generation sequencing for unbiased pathogen identification. Respiratory samples collected in Thailand, Philippines, Bhutan, and Nepal, that were negative by several molecular and immunofluorescence assays, underwent viral cultivation. Samples which demonstrated cytopathic effect in culture (N = 121) were extracted and tested by Luminex xTAG respiratory viral panel (RVP) assay and deep sequencing by Roche 454 FLX Titanium system. Using RVP assay, 52 (43%) samples were positive for enterovirus or rhinovirus and another three were positive for respiratory syncytial virus B, parainfluenza 4, and adenovirus. Deep sequencing confirmed the Luminex assay results and identified additional viral pathogens. Human enteroviruses, including Enterovirus A type 71 and 12 types of Enterovirus B (EV-B) were identified from a hospital in Bangkok. Phylogenetic and recombination analysis showed high correlation of VP1 gene-based phylogeny with genome-wide phylogeny and the frequent genetic exchange among EV-B viruses. The high number and diversity of enteroviruses in the hospital in Bangkok suggests prevalent existence. The metagenomic approach used in our study enabled comprehensive diagnoses of respiratory viruses.

Detection of a divergent Parainfluenza 4 virus in an adult patient with influenza like illness using next-generation sequencing.

BACKGROUND: Human Parainfluenza viruses are a common cause of both upper and lower respiratory tract infections, particularly in children. Of the four Parainfluenza virus serotypes, Parainfluenza 4 is least well characterised from both the clinical, epidemiological and genetic perspectives. METHODS: Flocked nose or throat swabs from a previous study investigating viral prevalence in community-based adults suffering from influenza like illness were used as the basis for this study. Samples in which no virus was detected using a 16 viral respiratory pathogen real-time PCR panel were barcoded and pyrosequenced using the Roche 454 GS FLX Titanium chemistry. The sequences were analysed using the VirusHunter bioinformatic pipeline. Sanger sequencing was used to complete the detected Parainfluenza 4 coding region. RESULTS: A variant Parainfluenza 4 subtype b strain (QLD-01) was discovered in an otherwise healthy adult who presented with influenza like illness. Strain QLD-01 shared genomic similarities with both a and b subtypes. The extent of divergence of this genome from the 5 available whole Parainfluenza 4 genomes impacted the predicted binding efficiencies of the majority of published Parainfluenza 4 PCR assays. CONCLUSIONS: These findings further support a possible role for Parainfluenza 4 in the aetiology of adult respiratory disease within the community setting, and highlight the caution needed to be used in designing PCR assays from limited sequence information or in using proprietary commercial PCR assays.

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent "primer walking" combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

Epidemiology and clinical presentation of the four human parainfluenza virus types.

BACKGROUND: Human parainfluenza viruses (HPIVs) are important causes of upper respiratory tract illness (URTI) and lower respiratory tract illness (LRTI). To analyse epidemiologic and clinical characteristics of the four types of human parainfluenza viruses (HPIVs), patients with acute respiratory tract illness (ARTI) were studied in Guangzhou, southern China. METHODS: Throat swabs (n=4755) were collected and tested from children and adults with ARTI over a 26-month period, and 4447 of 4755 (93.5%) patients' clinical presentations were recorded for further analysis. RESULTS: Of 4755 patients tested, 178 (3.7%) were positive for HPIV. Ninety-nine (2.1%) samples were positive for HPIV-3, 58 (1.2%) for HPIV-1, 19 (0.4%) for HPIV-2 and 8 (0.2%) for HPIV-4. 160/178 (88.9%) HPIV-positive samples were from paediatric patients younger than 5 years old, but no infant under one month of age was HPIV positive. Seasonal peaks of HPIV-3 and HPIV-1 occurred as autumn turned to winter and summer turned to autumn. HPIV-2 and HPIV-4 were detected less frequently, and their frequency of isolation increased when the frequency of HPIV-3 and HPIV-1 declined. HPIV infection led to a wide spectrum of symptoms, and more "hoarseness" (p=0.015), "abnormal pulmonary breathing sound" (p<0.001), "dyspnoea" (p<0.001), "pneumonia" (p=0.01), and "diarrhoea" (p<0.001) presented in HPIV-positive patients than HPIV-negative patients. 10/10 (100%) HPIV-positive adult patients (≥14 years old) presented with systemic influenza-like symptoms, while 90/164 (54.9%) HPIV-positive paediatric patients (<14 years old) presented with these symptoms (p=0.005). The only significant difference in clinical presentation between HPIV types was "Expectoration" (p<0.001). Co-infections were common, with 33.3%-63.2% of samples positive for the four HPIV types also testing positive for other respiratory pathogens. However, no significant differences were seen in clinical presentation between patients solely infected with HPIV and patients co-infected with HPIV and other respiratory pathogens. CONCLUSIONS: HPIV infection led to a wide spectrum of symptoms, and similar clinical manifestations were found in the patients with four different types of HPIVs. The study suggested pathogenic activity of HPIV in gastrointestinal illness. The clinical presentation of HPIV infection may differ by patient age.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008.

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences.

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.

Clinical and molecular epidemiology of human parainfluenza virus 4 infections in hong kong: subtype 4B as common as subtype 4A.

In this 1-year study, 35 (1.2%) of 2,912 nasopharyngeal aspirates were positive for human parainfluenza virus 4 (HPIV4) by reverse transcription-PCR. Patients with HPIV4 infection were mainly young children and immunocompromised adults. In contrast to the reported predominance of HPIV4A infection, molecular subtyping revealed that 15 (44%) cases were caused by HPIV4B.

Aetiology of influenza-like illness in adults includes parainfluenzavirus type 4.

Influenza viruses cause significant morbidity and mortality in adults each winter. At the same time, other respiratory viruses circulate and cause respiratory illness with influenza-like symptoms. Human respiratory syncytial virus (HRSV), human parainfluenza viruses (HPIV) and human metapneumovirus have all been associated with morbidity and mortality in adults, including nosocomial infections. This study evaluated 154 respiratory specimens collected from adults with influenza-like/acute respiratory illness (ILI) seen at the Edward Hines Jr VA Hospital, Hines, IL, USA, during two successive winters, 1998-1999 and 1999-2000. The samples were tested for ten viruses in two nested multiplex RT-PCRs. One to three respiratory viruses were detected in 68 % of the samples. As expected, influenza A virus (FLU-A) infections were most common (50 % of the samples), followed by HRSV-A (16 %). Surprisingly, HPIV-4 infections (5.8 %) were the third most prevalent. Mixed infections were also relatively common (11 %). When present, HPIV infections were approximately three times more likely to be included in a mixed infection than FLU-A or HRSV. Mixed infections and HPIV-4 are likely to be missed using rapid diagnostic tests. This study confirms that ILI in adults and the elderly can be caused by HRSV and HPIVs, including HPIV-4, which co-circulate with FLU-A.

Respiratory viruses in HIV-infected patients with suspected respiratory opportunistic infection.

OBJECTIVE: To assess the incidence and epidemiological pattern of respiratory viruses in HIV-infected patients and to evaluate their potential clinical impact. DESIGN AND METHODS: A prospective population-based cohort study was conducted at three Swiss university hospitals. Study participants were HIV-infected patients who underwent a bronchoalveolar lavage to rule out an opportunistic event. All bronchoalveolar lavage specimens were screened using a set of real-time reverse transcriptase-polymerase chain reaction assays targeting 17 different respiratory viruses. RESULTS: Between November 2003 and November 2006, 59 bronchoalveolar episodes from 55 HIV-infected patients were analysed. Eleven of 59 episodes (18.6%) were positive for at least one respiratory virus. Coronavirus OC43 was identified in three cases (27.3%) followed by influenza A in two (18.2%). Parainfluenza virus (PIV) 2, PIV 3, PIV 4, bocavirus, human rhinovirus A and human metapneumovirus were each identified in one case (9%). In the majority of these cases (63.6%) no other concomitant microorganism was isolated. CONCLUSIONS: Clinical investigation of respiratory viral infections in HIV-infected patients should not be restricted to prototype viruses and also need to target all the different family of viruses as it seems likely that these viruses contribute to pulmonary complications and morbidity in this population.

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.

A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.