Torque Teno Virus in Nasopharyngeal Aspirate of Children With Respiratory Infections

Torque teno virus (TTV) is responsible for persistent infections and is considered a marker of immune function. The role of TTV as a facilitator of respiratory infections(RIs) is unknown. We aimed to estimate the prevalence of TTV in the nasopharyngeal aspirate (NPA) of hospitalized children with RIs and correlate them with outcomes and immune response. NPA was taken for testing 16 respiratory viruses by RT-polymerase chain reaction (PCR), TTV PCR, and immunological study. Sixty hospitalized children with an RI and 3 healthy control infants were included. A total of 51/60 patients had a positive common respiratory viral (CRV) identication. A total of 24/63 (38.1%) children were TTV+ and had other CRVs in 95.8% of cases vs 74.4% in TTV- (p=0.029). TTV+ patients tended to be older, have fever, and need PICU admission more often than TTV- patients. Abnormal chest X-ray was more frequent in the TTV+ patients, OR 2.6(95% CI:1.3-5.2). The genetic expression of laggrin (involved in epithelial barrier integrity) was lower in TTV+ patients; however, levels of laggrin in the NPA were increased. release. During as study, questionnaire following variables: age; sex; month of admission; clinical diagnosis; history of prematurity or underlying chronic diseases; need for oxygen therapy, evaluated via transcutaneous oxygen saturation; fever; maximum axillary temperature; presence of inltrates and/or atelectasis in chest X-rays; administration of antibiotic therapy; length of hospital stay; need for admission to a Pediatric Intensive Care Unit (PICU); total white blood cell count; serum C-reactive protein levels (mg/L); and blood culture results (in those cases for which such tests had been performed). As part our global study of a small number for non-infectious causes (e.g., vaccines, minor surgery) were included as


Introduction
Torque teno virus (TTV) is a prototype anellovirus, a small ubiquitous DNA virus responsible for persistent asymptomatic infections 1,2 . TTV is considered to be a marker of immunological status 3 . An increase in TTV replication has been observed in sepsis 4 , HIV infection 5,6,7 , untreated cancer 8 , bone marrow transplantation 9,10 , and solid organ transplantation 11,12,13 as a potential endogenous marker of immunosuppression. In terms of respiratory infections, TTV has been found in children with recurrent pneumonias, showing its potential to infect the respiratory tract 14 . It has also been associated with bronchopneumonia, and its role in asthma is under study 15,16 . As far as we know, there have been few studies of TTV in respiratory samples of children with respiratory tract infections, correlating its presence with the immunological state or its clinical evolution 15 . The role of TTV as a facilitator of respiratory infections or airway in ammation remains to be determined.
The objective of this study was to determine the prevalence of TTV in hospitalized children with respiratory infections, analyzing their nasopharyngeal aspirate samples and how viral detection correlates with individual clinical evolution. We also analyzed the innate immune response of the children who were positive for TTV.

Patients And Methods
This is a substudy of an ongoing prospective investigation of respiratory tract infections in children, approved by the Medical Ethics Committee of Carlos III Health Institute; Ethics Committee of Hospital La Paz and Ethics Committee of Hospital Severo Ochoa. All research was performed in accordance with regulations and the Declaration of Helsinki. Informed consent was obtained from all parents or legal guardians.

Clinical assessment
This was a multicenter study performed in Madrid, Spain, between January 2021 and June 2021. The study population was comprised of children younger than 5 years of age with acute respiratory infection (ARI) admitted to either of the participating hospitals, Severo Ochoa (Leganés) or La Paz University Hospital (Madrid). The exclusion criterion was a refusal to participate in the study. Patients were evaluated by an attending physician, and the patients' clinical characteristics were analyzed. During their hospital stay, and as part of the study, a physician completed a questionnaire with the following variables: age; sex; month of admission; clinical diagnosis; history of prematurity or underlying chronic diseases; need for oxygen therapy, evaluated via transcutaneous oxygen saturation; fever; maximum axillary temperature; presence of in ltrates and/or atelectasis in chest X-rays; administration of antibiotic therapy; length of hospital stay; need for admission to a Pediatric Intensive Care Unit (PICU); total white blood cell count; serum C-reactive protein levels (mg/L); and blood culture results (in those cases for which such tests had been performed). As part of our global study of respiratory infections, a small number of healthy children attended as outpatients for non-infectious causes (e.g., vaccines, minor surgery) were included as controls.
Acute expiratory wheezing was considered to be bronchiolitis when it occurred for the rst time in children aged younger than 2 years, following the classic criteria of McConnochie 17 . All other episodes of acute expiratory wheezing were considered to be recurrent wheezing. Laryngotracheobronchitis was associated with inspiratory stridor and wheezing. Laryngitis was associated with inspiratory stridor without wheezing. Cases with both focal in ltrates and consolidation in chest X-rays were, in the absence of wheezing, classi ed as pneumonia. However, those cases with wheezing were classi ed as bronchiolitis or recurrent wheezing as appropriate.

Viral studies
Specimens consisted of nasopharyngeal aspirates (NPAs) that were obtained from each patient at admission. NPAs and nasopharyngeal swabs were sent for virological investigation to the Respiratory Viruses and In uenza Unit at the National Center for Microbiology (ISCIII), Madrid, Spain. Samples were stored at 4 ºC in a refrigerator and were processed within 24 hours after collection. Upon reception, 3 aliquots were prepared and stored at −80 ºC. Both the reception and the NPA sample processing areas are separated from those de ned as working areas.
RNA and DNA from 200-µl aliquots of NPA were extracted with the QIAamp Mini Elute Virus spin kit in an automated extractor (QIAcube, Qiagen, Valencia, CA, USA). Detection of respiratory virus was performed by 4 independent multiplex reverse transcription-polymerase chain reaction (RT-PCR) assays. The rst assay detected In uenza A, B, and C viruses; the second was used to detect parain uenza viruses 1 to 4, human rhinoviruses (HRVs), and enteroviruses; and the third assay detected the presence of respiratory syncytial virus (RSV) types A and B, human metapneumovirus, human bocavirus, and human adenoviruses (HAdVs). These 3 assays were real time multiplex RT-PCRs and used the SuperScript™ III Platinum® One-Step Quantitative RT-PCR System (Invitrogen). A fourth multiplex RT-PCR was used for investigation of human coronavirus (HCoV), using generic primers that were able to detect both alpha and beta coronavirus. Typing of HCoV was performed using a reverse speci c primer for detection of HCoV 229E, HCoV NL63, HCoV OC43, and HCoV HKU1. Primers and Taqman probes for the 3 independent multiplex real time RT-PCRs were based on previously published designs by our group 18 , and the HCoV primers are available on request.
Torque teno virus study A generic PCR assay was designed for the detection of the TTV diverse group. Previous studies had described a variation in the rate of positive detection depending on the target site of ampli cation in the PCR. Given that the untranslated regions (UTRs) of the genome are more conserved as compared to the open reading frames, primers designed in the UTR will cross-match a large number of genotypes and increase the rate of detection 19,20 .
This method involved cycling consisting of a denaturation cycle of 95 ºC for 5 minutes, followed by 40 cycles with a denaturation step of 30 seconds at 95 ºC, an annealing step of 30 seconds at 57 ºC and an extension step of 1 minute at 72 ºC. The nal extension step was 72 ºC for 5 minutes.
Immunological study A portion of each NPA sample was centrifuged to obtain the cellular pellet and supernatant. Samples with mucus were ltered with a 40-µm nylon lter. The pellet was resuspended in 0.7 mL Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and frozen at −80 ºC. The supernatants were also frozen at −80 ºC.

Statistical analysis
The descriptive data were expressed as mean and rst and third quartile (interquartile range [IQR]) for the continuous variables, and through counts and percentages for the categorical variables.
The continuous variables that followed a normal distribution were compared using a one-way analysis of variance with Bonferroni correction, or through T tests. When the distribution was not normal, we used the Mann-Whitney U test or Kruskal-Wallis test with Dunn correction. The categorical variables were compared using a chi-squared test or Fisher's exact test, and results were expressed as odds ratios (ORs).
P-values <0.05 were considered statistically signi cant, and con dence intervals were calculated at 95% for all the estimations. The analyses were performed using SPSS software (version 21; SPSS Inc, Chicago, IL, USA) and with Graph-Pad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

Results
The study population consisted of 60 hospitalized children with a diagnosis of respiratory infection and 3 healthy control infants. A total of 51/60 (85%) patients had a positive common respiratory virus (CRV) identi cation, and 24 (47%) were coinfected with more than one virus. The median age was 11.8 months (IQR 1.4-23.5), 66.7% were male, and 9 patients had been born preterm (14.3%). The children were mainly recruited (75%) in April and May of 2021, coinciding in this pandemic year with a higher incidence of RSV respiratory infections during those months. The most commonly identi ed viruses were HRV and RSV in the same proportion (27/63; 42.9%), followed by human bocavirus (14; 32.2%) and HAdV (9; 14.3%), 2 parain uenza, 1 HCoV, and 1 metapneumovirus.
TTV was identi ed in a total of 24/63 (38.1%) children, 23 in the case group and one in the healthy control group, who interestingly also had an asymptomatic HRV detection. No respiratory virus was detected in the two remaining children in the control group.

Comparison between TTV-positive and -negative patients
The 24 positive TTV patients were compared with the 39 TTV-negative children. The clinical data are shown in Table 1. Viral coinfection with another CRV was present in 95.8% (23/24) of the TTV-positive patients vs 74.4% (29/39) in the TTV-negative patients, p=0.029. No speci c CRV was signi cantly associated with the presence of TTV. The two positive parain uenza cases were associated with detection of TTV in NPA. Viral CRV coinfections were not associated with TTV presence.
The TTV-positive patients tended to be older, to have fever, and to need more frequent PICU admission than the negative ones, although the differences did not reach statistical signi cance. The TTV-positive children were also more likely to have radiological abnormalities, OR 2.6 (95% CI 1.3-5.2), p=0.030.

Evaluation of the immune response and barrier integrity in children with and without TTV infection
An aliquot of NPA was available in 25 patients, 12 with positive TTV (one was a healthy control) and another 13 with negative TTV (two were healthy controls).

Discussion
The prevalence of TTV infection in children with ARI was high in our study, reaching 38% of cases. It was associated with an infection by another respiratory virus in practically all cases. The children with detection of TTV in NPA more frequently had pneumonia on radiography, and a clinical course with fever and frequent admission to the PICU. Our results appear to indicate that TTV infection is a marker of impaired immune response and a facilitator of respiratory infections and greater severity. The study of the immune response of our patients has shown that TTV+ children have a higher abundance of the laggrin protein in NPA, with a lower gene expression. This result could mean that there is an alteration of the epithelial barrier in the TTV+ group, which is associated with liberation of this molecule to the supernatant of their NPA. Filaggrin is a protein involved in the integrity of the epithelial barrier and could play a role in allowing or favoring these respiratory infections 22 .
Chronic TTV infections have been reported in healthy individuals, and colonization is considered to begin very early in life, possibly transplacental in some cases, or in the family environment by the fecal-oral route 23,24 . Colonization increases with age. Although the prevalence of TTV in blood samples has been more often studied than in respiratory secretions, the respiratory route is considered to be a frequent path of dissemination and contagion 15 . The role of TTV in pediatric respiratory infections is poorly understood. Maggi F et al 15 , in the most important study in this regard, found that the presence of TTV was associated with bronchopneumonia and other respiratory viral infections, similarly to our study. They hypothesized that coinfection could increase the severity of other respiratory infections. Our study supports this hypothesis, and we observed that our TTV+ patients had coinfection with other CRVs and a more severe clinical outcome.
To study the respiratory secretions of these children in terms of their immune response is tempting, to better understand the pathophysiology of this association. The TTV+ patients had higher expression of TLR3 and IL33 in the NPA. Although they were not statistically signi cant results, this lack of signi cance might be due to the small sample analyzed; however, it suggests that in the group infected by TTV there is a higher detection of genes upregulated by viral infection. The increased expression of TLR3 and IL-33 in NPA has been previously described by our group, primarily in children with bronchiolitis, and this was the main diagnosis in our cohort 25 . They have also been overexpressed in animal models of asthma exacerbation 26 . Some studies have shown that viruses like RSV or HRV induce IL-33 synthesis by damaging epithelium in pulmonary diseases 27 .
The most important nding is that the frequency of TTV+ children that expressed laggrin mRNA was lower than in the TTV-, and that the laggrin protein was nevertheless increased in NPA from the TTV+. Filaggrin is a protein involved in the integrity of the epithelial barrier and its mutations have been associated with atopic dermatitis or ichthyosis 28, 29 . In our TTV+ patients, laggrin is likely underexpressed, and the increase of laggrin in NPA is part of the ongoing repair mechanism to maintain barrier function, triggered to respond to the epithelial damage caused by viral infection. Filaggrin release from keratohyalin granules into the keratinocyte cytoplasm is a main event in the corni cation process, and it is critical for skin barrier function. The increase of laggrin observed in the supernatant might be explained by damage to the cellular integrity and the consequent liberation of intracellular laggrin to the extracellular medium 30 .
Whether the presence of TTV in respiratory samples is a marker of a de cient immune response or is a consequence of it is di cult to elucidate. We cannot rule out that the previous presence of chronic TTV infection predisposes patients to infection by other CRVs and to a greater severity.
On the other hand, the association of altered laggrin with the possible development of asthma, as well as increased Th-2 immune response in our patients who clinically had a diagnosis of bronchiolitis or recurrent wheezing, suggest that the presence of TTV infection could have a role in respiratory diseases. Several studies have suggested that TTV plays a role in the development and/or exacerbation of respiratory diseases in childhood such as asthma 31 . It has been postulated that TTV has a role in respiratory dysfunction, either alone or synergistically with other viruses, and could act as an enhancer of in ammation systemically or at speci c body sites, such as the upper and lower airways 32 .
A limitation of our study is that it was performed during a pandemic. Thus, and as has been previously mentioned, the sample size was small, and although plausible, it does not allow for rm conclusions to be drawn about the immune response. However, it provides a comprehensive view of TTV respiratory infection, which has been little studied so far, in a homogeneous population of infants.
In conclusion, TTV infection is common in children with viral respiratory infections and could be associated with pneumonia and greater severity, as well as an alteration in the epithelial barrier due to low laggrin gene expression. Larger prospective studies will be able to unravel whether TTV favors respiratory viral infections or is a marker of impaired immune response.
* Negative TTV was a protective factor for CRV identi cation. Signi cant differences are in bold Figures Figure 1 Filaggrin expression is more frequent in infants without TTV, whereas its quantity in the supernatant is higher in the TTV+ group. (A) Frequency (%) of infants expressing the selected mRNA transcripts (TLR3,