








key: cord-336636-xgfw21hk
authors: Spezia, Pietro Giorgio; Macera, Lisa; Mazzetti, Paola; Curcio, Michele; Biagini, Chiara; Sciandra, Ilaria; Turriziani, Ombretta; Lai, Michele; Antonelli, Guido; Pistello, Mauro; Maggi, Fabrizio
title: Redondovirus DNA in human respiratory samples
date: 2020-08-15
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104586
sha: 
doc_id: 336636
cord_uid: xgfw21hk

Abstract Background Redondovirus (ReDoV) is a recently discovered circular, Rep-encoding single-stranded DNA (CRESS-DNA) virus in humans. Its pathogenesis and clinical associations are still completely unknown. Methods The presence of ReDoV DNA was investigated in biological specimens of 543 Italian subjects by in-house developed PCR assays. Results The overall ReDoV prevalence was about 4% (23 of 543 samples). The virus was detected in 22 of 209 (11 %) respiratory samples. One stool sample was also ReDoV positive. Viral DNA was not found in blood samples from immunocompetent and immunosuppressed subjects and cerebrospinal fluids from patients with neurological diseases. Genomic nucleotide differences were detected among the ReDoV isolates by sequencing a 582-nucleotide fragment of the capsid gene of the viral genome. Conclusions The results demonstrate that ReDoV is mainly present in the respiratory tract of infected people. Further investigations are needed to reveal possible clinical implications of this new CRESS-DNA virus in humans.

While attempting to study human virome in bronchoalveolar lavage (BAL) samples of lung transplant patients [1, 2] , Abbas and colleagues identified sequence reads that aligned with low-coverage to a poorly characterized circovirus, named porcine stool-associated circular virus-5 (PoSCV-5) [3] .

Further genomic characterization of these reads revealed that they belonged to a novel virus having a single, closed molecule of circular DNA approximately 3,000 nucleotides (nt) in length called Redondovirus (ReDoV) [4] . It was proposed as the second most prevalent eukaryotic virus, after anelloviruses, in human respiratory samples from viral metagenomics studies. The circumstance that the genomic organization and homology of ReDoV differ from that of other known circular, singlestranded DNA viruses [including those belonging to the group of circular Rep-encoding singlestranded DNA (CRESS) viruses] [5, 6] has suggested that ReDoV is the first member of the new viral family, named Redondoviridae [4, 7] . To date, 22 ReDoV genomes have been completely sequenced [4, 8] and, based on 50% Rep protein identity [9] [10] [11] , grouped in two species highly prevalent in the respiratory tract: Vientovirus and Brisavirus [4] .

The ReDoV genome contains two ambisense major open reading frames (ORF) encoding capsid and Rep proteins [449-531 and 334-363 amino acids, respectively] [4] . The capsid protein, like that of other single-stranded DNA viruses [5, 6, 12] , contains a basic amino terminus, while the Rep protein has two domains, similar to many small DNA and RNA viruses [13, 14] . Surprisingly, the capsid protein is more conserved than Rep protein among ReDoV sequences, being the range of amino acid identities (67.5-99.6% and 36.6-99.7%, respectively). All ReDoV genomes also contain a third ORF (ORF3) overlapping the capsid gene. Epidemiology, biological properties, and pathogenic potential of ReDoV are still completely understood. Abbas and colleagues [4] have investigated ReDoV prevalence by re-analyzing for homology to the virus metagenomics sequence data of 7,581 samples from 173 datasets covering 51 organisms or environments and 18 human body sites or fluids. ReDoV sequences were detected only in human samples, from oral cavity (3.8%), lung (3.3%), nasopharynx J o u r n a l P r e -p r o o f (0.95%), and gut (0.59%). The presence of ReDoV was also tested in oropharyngeal swabs from 129 individuals by real-time PCR. ReDoV DNA was detected in 12%, at levels that in critically diseased patients were 10 4 -fold higher than in healthy individuals. Additionally, consecutive samples from some subjects obtained at later times for 2-3 weeks remained ReDoV positive, suggesting the persistence of virus infection. These observations suggest that ReDoV is not part of the normal oral and/or respiratory microflora of humans, differently to other circular single-stranded DNA viruses [5, 15] and that its infection might be involved in clinically relevant disorders. 

A total of 543 human biological specimens were studied. Specimens had been submitted to our laboratory by local hospitals where they were processed for routine virological analysis. The study was run after ethical approval from Comitato Etico di Area Vasta Nord-Ovest (protocol numbers 39238, and 63409). The most specimens (n. 443) were obtained from diseased patients: 209 respiratory specimens (151 nasopharyngeal swabs, 36 sputum samples, and 22 pharyngeal swabs obtained between April and July 2019) from individuals with acute and chronic respiratory diseases, 79 whole blood samples from transplant recipients, 105 stools from individuals with gastroenteric illness, and 50 cerebrospinal fluids from neurological patients. The remaining 100 plasma samples were obtained from healthy blood donors. All the respiratory specimens were submitted to systematic testing for common respiratory virus detection by commercial real-time PCR assays, according to the manufacturer's instructions.

Viral DNA was extracted from 200 µl of samples by using the QIAamp DNA Mini kit (QIAGEN, Chatsworth, CA) manually or associated with a QIAsymphony SP/AS instrument. Extracted DNA was amplified with two different PCR protocols, both developed in our laboratories and targeting the capsid gene of the viral genome.

The first amplification was performed by a semiquantitative one-step real-time PCR based on SYBR-Green PCR (SY-PCR) coupled with a melting temperature analysis of 90 nucleotide-length fragment. experiments, and the coefficient of variation was calculated. The differences between input (830,000 copies) and calculated copy numbers were small, the latter ranging between 733,000 and 862,000

with the maximum inter-assay variation lower than 0.1 Log, thus indicating good reproducibility of the SY-PCR assay. The lower detection limit of the procedure was measured by testing serial dilutions of a known concentration of a recombinant plasmid and was found to be of 10 copies. Table 2) .

Only one stool specimen resulted positive for ReDoV. This was from a 60-year old woman receiving hematopoietic stem cell transplantation (HSCT) for a history of lymphoma. This patient's stool sample was also positive for rotavirus antigen.

Sequencing was limited to 10 amplicons obtained from independent PCR runs, confirming the detection of ReDoV DNA, and phylogenetic analysis showed that all the isolates were related to the previously published strains (Figure 1) . Most of them were very closely related (overall mean distance: 0.104; range: 0.000-0.295). Again, our isolates differed little from the ones already in GenBank (Figure 1 ).

The recent discovery of ReDoV inspired this retrospective study on biological specimens collected from diseased patients and healthy blood donors. [11, 17] .

In this study, the majority of virus-positive patients had more severe respiratory diseases, and most of them yielded no other common respiratory viruses and/or microbial agents that might have been responsible for the clinical forms from which the patients suffered. While the number of patients studied here is too small to be definitive regarding the association of ReDoV with severe respiratory tract disease, the observation of particularly higher ReDoV levels in critically ill patients than in healthy subjects [4] encourages to pursue the observation furtherly focusing on expanding the sample set and on specific pulmonary disease cohorts (e.g. asthma, cystic fibrosis, etc). After all, the finding suggests that ReDoV could be not a frequent commensal virus inhabiting the respiratory tract in the absence of symptomatic disease. Again, the follow-up of ReDoV positive patients will be important J o u r n a l P r e -p r o o f for understanding if the virus remains consistently persistent or is able only to give short-lasting acute infections in the infected host.

Of interest is the demonstration of ReDoV DNA in the feces of one subject. The finding might indicate that, similar to other respiratory viruses [18, 19] , ReDoV may not remain restricted to the respiratory tract. It is noteworthy that the subject in whom the observation was done receiving HSCT, suggesting that an immunosuppressed status of an infected host could favor this event. However, further studies will be needed to establish whether ReDoV can represent a further agent of viral enteritis or can just be excreted with the stools similar to other viruses primarily infecting the respiratory tract. Again, the short PCR fragment sequenced does not allow us to classify the ReDoV DNAs in one of the two species in which the virus has been genetically characterized. Sequencing of near full-length or full-length genomes will be necessary for better exploring the genetic variability of ReDoV and for improving its phylogenetic classification. 

We declare that we have no conflicts of interest.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

I declare that the authors haven't any financial or personal relationship with other people or organizations that could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.

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The aminoacidic (A) and nt (B) trees based on a 582-bp segment from the capsid gene of the viral genome was obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using Jones-Thornton-Taylor (JTT) and Maximum Composite Likelihood (MCL) models, respectively. Bootstrap resampling (1,000 replicates) was used to test the robustness of the trees. The tree was drawn by using MEGA X program (version 10.0.5). ReDoV sequences from the present study obtained by respiratory and stool samples are indicated by solid and open circles, respectively. The 22 sequences of ReDoV

The Porcine stool-associated circular virus/BEL/15V010 isolate 15V010 (accession number KY214434) was used as the outgroup. The bar represents the number of substitutions per site

J o u r n a l P r e -p r o o f

