key: cord-0703295-6d0yvixw
authors: Prasetyo, Afiono Agung; Desyardi, Martinus Nuherwan; Tanamas, Jimmy; Suradi,; Reviono,; Harsini,; Kageyama, Seiji; Chikumi, Hiroki; Shimizu, Eiji
title: Respiratory Viruses and Torque Teno Virus in Adults with Acute Respiratory Infections
date: 2015-02-10
journal: Intervirology
DOI: 10.1159/000369211
sha: c138634bcadb1b077d8303f8f7618aa39337d979
doc_id: 703295
cord_uid: 6d0yvixw

OBJECTIVE: To define the molecular epidemiology of respiratory viral infections in adult patients. METHODS: Nasal and throat swabs were collected from all adult patients with influenza-like illness (ILI), acute respiratory infection (ARI), or severe ARI (SARI) admitted to a tertiary hospital in Surakarta, Indonesia, between March 2010 and April 2011 and analyzed for 19 respiratory viruses and for torque teno virus (TTV) and human gyrovirus (HGyV). RESULTS: Respiratory viruses were detected in 61.3% of the subjects, most of whom had ARI (90.8%, OR = 11.39), were hospitalized (96.9%, OR = 22.31), had asthma exacerbation (90.9%, OR = 8.67), and/or had pneumonia (80%, OR = 4.0). Human rhinovirus (HRV) A43 predominated. Influenza A H3N2, human metapneumovirus (HMPV) subtypes A1 and A2, the influenza B virus, human adenovirus B, and human coronavirus OC43 were also detected. All respiratory viruses were detected in the transition month between the rainy and dry seasons. No mixed respiratory virus infection was found. Coinfections of the influenza A H3N2 virus with TTV, HMPV with TTV, HRV with TTV, and human parainfluenza virus-3 with TTV were found in 4.7, 2.8, 19.8, and 0.9% of the samples, respectively. CONCLUSIONS: This study highlights the need to perform routine detection of respiratory viruses in adults hospitalized with ARI, asthma exacerbation, and/or pneumonia.

Adequate molecular epidemiology databases of infec human gyrovirus (HGyV). Results: Respiratory viruses were tious agents are important for infection prevention and detected in 61.3% of the subjects, most of whom had ARI treatment programs. However, an adequate database of (90.8%, OR = 11.39), were hospitalized (96.9%, OR = 22.31), that type, particularly one that includes the respiratory vihad asthma exacerbation (90.9%, OR = 8.67), and/or had ruses, is not available in Indonesia, a developing country pneumonia (80%, OR = 4.0). Human rhinovirus (HRV) A43 located in Southeast Asia with a tropical climate and a large predominated. Influenza A H3N2, human metapneumovirus population (the fourth most populous country; http:// (HMPV) subtypes A1 and A2, the influenza B virus, human www.indonesia.go.id/en/indonesia-glance/geography adenovirus B, and human coronavirus OC43 were also de-indonesia). There is a lack of information concerning both tected. All respiratory viruses were detected in the transition the prevalence of respiratory virus infections and the viral month between the rainy and dry seasons. No mixed respira-genotypes circulating throughout Indonesia. The absence tory virus infection was found. Coinfections of the influenza of this information is a barrier to the development of ap propriate public health interventions, including immuni zation policies and health protection measures. Respira tory virus incidence and prevalence data throughout Indonesia are also very limited and have not been well doc umented. Only a few reports are available [1] [2] [3] [4] [5] [6] that dem onstrate the presence of influenza viruses in patient popu lations randomly selected from various studies. The pre sentation of respiratory syncytial virus (RSV) has been reported in children [7-10] but not in adults. There is no information about the presentation of other respiratory vi ruses, including their seasonal epidemic patterns and their association with specific clinical findings. Prevalence in formation is also required to direct approaches for secur ing appropriate clinical services for infected patients. Moreover, there are few data about respiratory viral infec tions in adults with acute respiratory infections (ARI) in tropical climates, especially in Southeast Asia, because most studies have been performed in children. The preva lence, clinical profile, and epidemiology of respiratory vi ruses in adults are different from those in young people [11] . Additionally, the relationship between the geograph ical distribution, season, and respiratory virus species is not fully understood in adults. Consequently, there is a clear need to further study the prevalence of respiratory viruses in adults with ARI. Based on these conditions, a cross-sectional study of adults with influenza-like illness (ILI), ARI, or severe ARI (SARI) was performed at the Dr. Moewardi General Hospital, a tertiary hospital in Surakar ta, Central Java, Indonesia, to determine the prevalence of a select group of human respiratory viruses, including the influenza A virus, the influenza A H1 virus, the influenza A H3 virus, the influenza A H5 virus, the influenza B virus, human parainfluenza virus (HPIV)-1, HPIV-2, HPIV-3, HPIV-4, RSV A, RSV B, human rhinovirus (HRV), entero virus (EV), human coronavirus (HCoV)-OC43, HCoV 229E, severe acute respiratory syndrome coronavirus (SARS-CoV), human metapneumovirus (HMPV), human bocavirus (HBoV), and adenovirus. This study aimed to describe the viral genotypes, evaluate the epidemic season ality, and determine whether specific characteristics were associated with these infections. The clinical presentation and coinfection with torque teno virus (TTV) and human gyrovirus (HGyV) were also investigated.

This study was performed from March, 2010 through April, 2011 at the Dr. Moewardi General Hospital in Surakarta, Central Java, Indonesia, the most densely populated city in Central Java and the eighth most densely populated city in Indonesia [12] . All adult patients admitted to the Department of Pulmonology of the Dr. Moewardi General Hospital with ILI, ARI, or SARI, according to the World Health Organization (WHO) case definition [13] , were enrolled into this study. Patients with underlying diseases (HIV/AIDS, diabetes mellitus, tuberculosis, alcoholism, chronic kidney disease, chronic liver disease, asplenia, leukopenia, cavitary infiltrates, pleural effusion, and sepsis) were excluded. Approval was obtained from the institutional ethical committee review boards of the Faculty of Medicine of Sebelas Maret University and the Dr. Moewardi General Hospital. Written informed consent was obtained from all individuals participating in this study. Up per respiratory (nasal and throat swabs) specimens were collected from each participant after informed consent had been obtained. The collected specimens were placed into viral transport media (BD Universal Viral Transport, Sparks, Md., USA) and kept at 4° during transportation. The clinical data were obtained from the patient medical records. All procedures were conducted according to the principles of the Declaration of Helsinki.

Both viral RNA and DNA were extracted on the same day of collection using a PureLink Viral RNA/DNA Kit (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instruc tions. The nucleic acids were then aliquoted, and one aliquot was reverse-transcribed using a Superscript III First-Strand cDNA Synthesis SuperMix Kit with random hexamers (Invitrogen). All DNA and cDNA were used immediately for nested multiplex PCR for 19 respiratory viruses as described previously [14] . Briefly, each nested multiplex PCR assay detected several pathogens: group 1 contained influenza A-and B-specific primers and subtype H1N1-, H3N2-, and H5N1-specific primers; group 2 contained primers for the parainfluenza viruses (PIV-1, PIV-2, PIV-3, PIV-4a, and PIV 4b); group 3 contained primers for RSV A, RSV B, HRV, and EV; group 4 contained primers for HCoV-OC43, HCoV-229E, SARS-CoV, and HMPV, and group 5 contained primers for HBoV, ad enovirus 1, adenovirus 2, adenovirus 3, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. Molecular detection was per formed by PCR using a AmpliTaq Gold ® 360 DNA Polymerase Kit (Invitrogen). Internal amplification controls were included in each molecular assay to exclude false-negative results. The correspond ing positive controls and one negative control (sterile water) sam ple were included for each group simultaneously. To prevent PCR contamination, the reagent preparation, sample processing, and nested PCR assays were performed in rooms separate from where the amplified products were analyzed. Aerosol-resistant pipette tips were used throughout the assays. The PCR products were sub jected to electrophoresis in 2% agarose gels, stained with ethidium bromide, and visualized under ultraviolet illumination. All sam ples were tested at least twice.

The pathological role of TTV in respiratory diseases remains poorly understood; therefore, all respiratory samples were also tested for TTV DNA by nested PCR amplification of a conserved region of ORF2, as described previously [15] . Internal amplifica tion controls were included in each molecular assay to exclude false-negative results. The corresponding positive controls and one negative control (sterile water) sample were included for each group simultaneously. PCR specificity was confirmed by sequenc ing of the amplicons. All samples were tested at least twice.

In 2011, a novel virus resembling chicken anemia virus (CAV) was reported and named HGyV [16] . The epidemiology, biologic properties, and pathogenic potential of HGyV remain poorly un derstood; therefore, all samples were also tested for HGyV DNA using a previously published nested PCR method [17] . Internal amplification controls were included in each molecular assay to exclude false-negative results. The corresponding positive controls and one negative control (sterile water) sample were included for each group simultaneously. PCR specificity was confirmed by se quencing the amplicons. All samples were tested at least twice.

The PCR products were purified from agarose gels, and nu cleotide sequencing was performed in both orientations using the inner primers from the nested PCR assays. The sequences were then submitted to the BLAST program to check their similarity to related strains deposited in GenBank/EMBL/DDBJ. The refer ence strains with the highest homology score to each analyzed strain were retrieved from the GenBank/EMBL/DDJB databases and aligned with the test sequences. All sequences of the selected respiratory viruses that were isolated in Indonesia and deposited in GenBank were also included in the alignment analysis for each tested sequence. However, with respect to the respiratory viruses studied here, only 4 Influenza A HA gene sequences from Indonesia that were deposited in GenBank could be found. The frequency of nucleotide substitution at each base was estimated using the Kimura 2-parameter method. A phylogenetic tree was constructed via the neighbor-joining method, and its reliability was estimated by 1,000 bootstrap replicates. The phylogenetic tree was constructed using the MEGA version 5 software package [18] .

Statistical analyses were performed using SPSS version 20 soft ware (SPSS, Chicago, Ill., USA), and 95% CI were used for all data analyses. OR were calculated according to a previously published method [19] .

The sequences described in this article have been deposited in GenBank/EMBL/DDBJ under accession numbers KC513508 to KC513572.

In total, 685 patients were admitted to the Department of Pulmonology of the Dr. Moewardi General Hospital in Surakarta, Indonesia, during the study period. Based on the WHO case definition of ILI, ARI, and SARI and the exclusion criteria, only 106 patients (63 men and 43 wom en) met the criteria; therefore, only 106 patients were en rolled into the present study.

The mean age of the study patients was 56.1 years (range 18-96). The mean BMI of the patients was 20.2 (range 12.9-31.1). Elevated respiratory rates of more than 20 breaths/min were found in 70.8% (75/106) of patients. Fe ver was found in 71.7% (76/106) of patients. Crackles were found in 44.3% (47/106) of patients, and wheezing was found in 38.7% (41/106) of patients. ILI, ARI, and SARI were initially diagnosed in 26.4% (28/106), 73.6% (78/106), and 0% (0/106) of patients, respectively. Based on the clin ical and radiological examinations [20] , pneumonia was found in 37.7% (40/106) of patients. Exacerbations of asth ma, chronic obstructive pulmonary disease (COPD), and bronchiectasis were found in 20.8% (22/106), 13.2% (14/106), and 2.8% (3/106) of patients, respectively. Nine teen (17.9%) patients had hypertension. Forty-eight (45.3%) patients had anemia, and 40 (37.7%) patients had leukocytosis. After careful medical examination of all pa tients, 19 patients (17.9%) were treated as outpatients, and 87 patients (82.1%) were hospitalized for a mean duration of 7.8 days (range 2-33). All patients received antibiotic therapy. One hundred three (97.2%) patients fully recov ered, whereas 3 patients died. None of the subjects enrolled into this study had ever received an influenza vaccination.

Respiratory viruses were detected in a total of 65 (61.3%) samples ( Values are presented as numbers.

1/32). All pneumonia patients who were positive for a re spiratory virus were hospitalized. M. pneumoniae and C. pneumoniae were not found in any sample. Unless oth erwise stated, no significant associations were detected between the presence of respiratory viruses and the clini cal features of the patients. Figure 1 illustrates the seasonality patterns for the re spiratory viruses detected in patients throughout the study period. HRV was detected almost every month and peaked during the 2010 dry season (August to October) and in the transition months from the dry season to the rainy season (November to December 2010). The influ enza A viruses, HMPV, adenoviruses, HPIV-3, and HCoV-OC43 were primarily detected during the transi tion months from the rainy season to the dry season (March to April) ( fig. 1 HMPV was detected only in samples derived from hospitalized patients with ARI (n = 8). One HMPV-pos itive sample was derived from a patient with asthma ex acerbation, and 1 was derived from a patient with COPD exacerbation; the remaining 6 HMPV-positive samples were not associated with any respiratory exacerbation ( ta ble 1 ). Six samples (15%, 6/40) derived from patients with pneumonia were positive for HMPV (OR = 5.65; 95% CI 1.081-29.508).

Based on the sequence from 432 nucleotides from the matrix protein (M) region (corresponding to nt 2279-2710 in HMPV-gz01, GenBank accession No. GQ153651), 4 HMPV isolates (IDSKAHMPV-3, IDSKAHMPV-4, ID SKAHMPV-6, and IDSKAHMPV-7) were clustered to gether with TN96-12, an isolate from the USA. Two HMPV isolates (IDSKAHMPV-1 and IDSKAHMPV-2) were closely related to SIN07-NTU442, an isolate from Singapore. IDSKAHMPV-5 shared 98.8% homology with an HMPV isolate from Japan (JPS03-240), and IDSKAHMPV-8 shared 99.8% homology with the HMPV gz01 isolate from Guangzhou, China. Two HMPV sub types were found: A1 (4/8) and A2 (4/8) ( fig. 3 ) amplicon were sequenced, corresponding to the viral 5UTR region (nt 168-461 in strain ATCC VR-1153, GenBank accession No. FJ445131). The HRV genotype A43 was found in 9 subjects, genotype A75 was found in 3 subjects, and genotype A19 was found in 2 subjects ( fig. 4 a) . HRV B RNA was successfully amplified by RT PCR from a total of 27 study samples, and 291 bp from each amplicon were sequenced, corresponding to the vi ral 5UTR region (nt 186-476 in strain ATCC VR-1182, GenBank accession No. FJ445153). The HRV genotype B3 was found in 7 subjects, genotype B37 was found in 3 subjects, genotype B6 was found in 7 subjects, genotype B14 was found in 4 subjects, genotype B72 was found in 5 subjects, and genotype B92 was found in 1 subject ( fig. 4 b) HPIV-3 was detected in one sample derived from a hospitalized ARI patient with asthma exacerbation ( table 1 ) . Based on a 717-bp segment of the hemaggluti nin-neuraminidase (HN) gene (corresponding to nt 7604-8320 in strain ZHYMgz01, GenBank accession No. EU326526), this isolate shared 97.4% homology with the HPIV-3 strain ZHYMgz01 from China.

TTV DNA was detected in 31.1% (33/106) of the sam ples, with 15.2% (5/33) taken from patients with ILI (OR = 0.09; 95% CI 0.026-0.293) and 84.8% (28/33) taken from patients with ARI (OR = 2.58; 95% CI 0.882-7.526). Three patients with samples positive for TTV DNA were not hospitalized, whereas 90.9% (30/33) of TTV-positive patients required hospitalization (OR = 2.81; 95% CI 0.757-10.403). Intriguingly, all ARI patients with samples positive for TTV DNA were hospitalized ( table 1 ) . Of the 30 samples coinfected with TTV and a human respiratory virus, 96.7% (29/30) were taken from hospitalized pa tients (OR = 58; 95% CI 2.56-1,313.926), and 27 were tak en from patients with an initial diagnosis of ARI (OR = 18; 95% CI 1.234-262.67). Five of the 11 (45.5%) samples infected with influenza A H3 were coinfected with TTV. Three of the 8 (37.5%) samples infected with HMPV were coinfected with TTV. Twenty-one of the 42 (50%) sam ples infected with HRV were coinfected with TTV. One HPIV-3-positive sample was also positive for TTV. Al most all hospitalized patients positive for TTV DNA were hospitalized for more than 3 days (90%, 27/30) ( tables 1 , 2 ). All samples were negative for HGyV.

Our study was conducted at a government-operated tertiary hospital that serves Surakarta and the surrounding area. One study revealed that Surakarta is the most dense ly populated city in Central Java and the eighth most densely populated city in Indonesia [12] . Surakarta has a tropical climate with a mean annual temperature of 30° and a constantly high relative humidity (>70%). It is low land, flat terrain at 105 m above sea level. Surakarta is cen trally located at the strategic paths connecting Semarang, Yogyakarta, and Surabaya, which is why Surakarta became an important business center for the surrounding district (http://www.surakarta.go.id/). Surakarta has 12 general hospitals; however, only the Dr. Moewardi General Hos pital acts as a government-operated tertiary hospital in Surakarta.

Respiratory viruses were detected in 61.3% of patients, with the majority being from hospitalized patients. Few reports exist on the role of viral respiratory infections in respiratory disease in adults. RSV, HMPV, and influenza have been reported to correlate with a significant number of hospitalizations in adults aged ≥ 50 years [21] . Picorna viruses have been reported as the most frequent virus causing infection among hospitalized adult patients with acute respiratory illness [22] . In the present study, HRV was detected most frequently, followed by the influenza A H3 virus, HMPV, the influenza B virus, adenovirus, HCoV-OC43, and HPIV-3. Our samples were mostly de rived from patients with ARI, suggesting that adults with ARI caused by a respiratory virus may be more likely to be hospitalized [23, 24] .

With regard to the seasonal patterns of the viruses, the seasonal distribution of the different viruses was variable and remarkable. HRV was detected almost every month and peaked during the dry season and during the transi tion month from the dry season to the rainy season, where as the influenza viruses, HMPV, adenovirus, HPIV-3, and HCoV-OC43 were detected primarily during the transi tion month from the rainy season to the dry season.

HRV was detected frequently in adult patients pre senting to the emergency department for respiratory dis tress and is associated with hospitalization, in line with our findings [25, 26] . However, in ILI patients, we pri marily detected HRV, followed by influenza A H3. This result is in contrast to a previous report from a tropical area [27, 28] , in which the influenza virus was the most common virus detected in adult patients with ILI in Ec uador or Maracay, Venezuela. Although Ecuador is also a tropical country, it has a high altitude (2,800 m) with a cool, dry environment and therefore has environmental conditions different from those of Surakarta. Maracay is a city near the Caribbean coast with mountains on its north side and therefore also has environmental condi tions different from those of Surakarta. Geographical and/or environmental conditions may affect the circula tion patterns of respiratory viruses [29, 30] .

Most patients with asthma exacerbation were positive for a respiratory virus, most often HRV, followed by the influenza A H3 virus, HMPV, and HPIV-3. Viral respira tory infections are already known as the most common cause of acute asthma exacerbation in both children and adults [31] .

In this study, respiratory viruses were detected in 80% of patients with pneumonia, which is a higher rate than previously reported [32, 33] . Moreover, all pneu monia patients infected with a respiratory virus were hospitalized. Pneumonia patients were most often in fected with HRV, followed by influenza A H3, HMPV, and influenza B. These results indicate that potential vi ral infections should be given more attention in adult pneumonia patients requiring hospitalization.

HRV was the most common viral agent detected in our study and was associated with ARI, hospitalization, asthma exacerbation, COPD exacerbation, and pneumo nia. HRV A43 predominated, followed by B3, B6, B72, B14, A75, B37, A19, B92, and C. We hypothesize that all species of HRV detected in our study are also spread among other regions in Indonesia, especially Java Island. At present, these findings represent the first and only molecular data in GenBank for an HRV isolate from Indonesia.

Although the influenza B virus was detected in only one sample derived from a hospitalized patient with ARI and pneumonia, the influenza A H3N2 virus was the sec ond most common viral agent detected in our study. Pre vious local studies have reported that influenza A H3N2 predominates in some sites in Indonesia [2, 5] , especially in ILI outpatients. At our study site, influenza A H3N2 was associated with ARI, hospitalization, asthma exacer bation, COPD exacerbation, and pneumonia. Based on our molecular analysis and comparisons with sequences from the GenBank/EMBL/DDBJ databases, the nucleo tide sequences of all influenza A H3N2 isolates in our study shared a higher homology with foreign strains than with any influenza A H3N2 virus isolated previously in Indonesia. This result suggests that foreign influenza A H3N2 strains are occasionally introduced into Indonesia.

We reported the circulation and molecular data for HMPV in Indonesia for the first time, to the best of our knowledge. HMPV was detected only in samples taken from hospitalized patients with ARI and was associated with pneumonia, which is consistent with previous find ings [34] . HMPV subtypes A1 and A2 were found circu lating in Surakarta. We also reported for the first time the circulation and molecular data for human adenovirus B, HCoV-OC43, and HPIV-3 in Indonesia, all detected from hospitalized ARI patients.

TTV was detected in nasal brushing samples of chil dren with recurrent or persistent pneumonia [35] . The TTV load was associated with airflow limitation within the peripheral airways in children with bronchiectasis or asthma [36, 37] . TTV may disrupt the mucociliary escala tor, similar to other respiratory viruses [35] ; however, its pathological mechanism remains poorly understood. In the present study, the detection of TTV was associated with ARI and hospitalization and, intriguingly, all TTVpositive ARI patients were hospitalized. Single infections with TTV were not detected in nasal and throat samples from patients with exacerbations of asthma, COPD, and bronchiectasis. In addition, single infections with TTV were not detected in nasal and throat samples from pa tients with pneumonia. However, 8 pneumonia samples were positive for coinfection with TTV and a respiratory virus. We also found that coinfection with TTV and a hu man respiratory virus was associated with the incidence of ARI. Additional studies are warranted to further inves tigate the association of age with the contribution of TTV to viral respiratory diseases, especially when coinfected with a respiratory virus.

HGyV, a novel virus resembling CAV, was detected in non-lesional skin specimens and in the blood of infected persons [16, 17] . HGyV could not be detected in bron choalveolar lavage fluid samples, nasopharyngeal aspi rates, or fecal samples from children [16] . However, CAV infects a wide range of cell types and has been associated with the worsening of pathologies caused by other viral and bacterial agents [38] [39] [40] . The absence of HGyV ge nomes in the respiratory specimens (nasal and throat swabs) derived from adults with ILI and ARI in the pres ent study supports the idea that HGyV may not replicate in the cells of the respiratory tract and may not be associ ated with respiratory diseases.

To the best of our knowledge, this report is the first molecular epidemiology study in Indonesia of respira tory viruses (influenza A H3, influenza B, HMPV, HRV, adenovirus, HCoV-OC43, and HPIV-3), especially in adult patients with acute upper respiratory infections. Moreover, prior to this study, there were only limited data about the molecular epidemiology profiles of respi References ratory viruses in Indonesia, and none of the data were derived from adult patients with ARI. HMPV, HRV, ad enovirus, HCoV-OC43, and HPIV-3 were also detected for the first time in Indonesia. However, our study had several limitations. We only tested for specific acute up per respiratory infection etiologies, including common human respiratory viruses and atypical bacteria; this limitation reflects a limited budget for etiological diag nosis, which is a common situation in hospitals in devel oping countries. The data collection was also limited to one tertiary hospital over a 1-year period due to the lim ited budget; therefore, the seasonal patterns for the vi ruses were derived from one season, and the number of study patients may have been insufficient to draw firm conclusions. Additional surveillance studies with larger sample sizes will be needed to confirm and extend our findings.

In our study, respiratory viruses were associated with ARI, asthma, pneumonia, and hospitalization, highlight ing the necessity for further studies of respiratory viruses in adults. Routine testing for respiratory viruses may also be warranted for adults who have been hospitalized with ARI, especially when they develop asthma exacerbation or pneumonia. The phylogenetic analysis suggested that foreign influenza A H3N2 strains have been occasionally introduced into Indonesia. Also, the molecular data of influenza B virus, HMPV, HRV, adenovirus, HCoV OC43, and HPIV-3 isolated in Indonesia are reported for the first time in the present report.

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