Demographic characteristics of the study
To investigate the association of ReDoV with human-associated viral diseases, the prevalence of ReDoV among individuals undergoing SARS-CoV-2 diagnostic testing was analyzed. The subjects consisted of 486 (66.48%) females (minimum age of 4 years and a maximum age of 83 years) and 245 (33.52%) males (ranging from 9 years to 99 years old) with a mean age of 37.97 ± 13.36 and 44.94 ± 17.06, respectively. For identifying any age-specific patterns, differences in susceptibility, or trends that might be present in different age groups and the ease of interpreting the results and understanding the prevalence of ReDoVs across different age groups, the subjects were divided into seven different age categories. According to the recorded documents of the subjects, the age distribution of this study illustrated a predominant representation in the 30–39 age group (29.82%), followed by individuals aged 40–49 (23.39%) and 20–29 (19.97%) (See Table 1).
Prevalence of SARS-CoV-2 among participants
The RT-PCR results of SARS-CoV-2 were obtained from the referrals’ records. As shown in Tables 1 and 131/731 (17.92%) were positive for SARS-CoV-2. Regarding vaccination status, a vast majority (97.13%) had received the COVID-19 vaccine, with the most common frequency being three times (45.69%). Sinopharm was the most prevalent vaccine type (19.84%) among those surveyed. Further information obtained by interviewing participants included gum or dental problems during their referral to the medical centers, clinical symptoms associated with COVID-19 disease (smelling or testing senses, fever, headache, bone ache, cough, chest pain, shortness of breath, and other flu-like symptoms, as well as underlying diseases and the type of medication (data are not shown), and influenza/COVID-19 vaccination data were. The presence of underlying diseases was noted in 14.77% of the population, while 14.23% reported taking medication. The underlying diseases included hypertension, diabetes, nephrotic syndrome, anemia, heart problems, arthritis rheumatoid (AR), asthma, stomach reflex, autism, blood fat, migraine, bronchitis, cancer, chemical veteran, endocarditis, fatty liver, gallstone, HIV, autoimmune disease, lung disease, allergy, hyperlipidemia, hyperprolactinemia, lupus, seizures, hyperthyroidism, hypothyroidism, intestinal bleeding, intestinal ulcer, Behçet’s disease, lymphoma, multiple sclerosis (MS), nervous and mental problems, osteoporosis, rheumatism, prostate cancer, myopathy, and psoriasis. To investigate the ReDoV genome further, the nasopharyngeal samples of the consent subjects were retrieved from the − 80 °C refrigerators, and viral genome extraction was performed, as described in the materials and methods section.
The ReDoV sequences obtained from the amplified samples were submitted to GenBank, and they will be available with accession numbers PP319424 to PP319438. A phylogenetic analysis was performed to measure further the genetic distance of the sequenced data with already known ReDoVs, and the data are depicted in Fig. 1.
Radial phylogram re-rooted with Mosquito VEM Anellovirus SDBVL A (NC_076121.1). The sequenced data of ReDoVs from the present study are colored. PP319426 and PP319425 are phylogenetically related to the human respiratory-associated Brisaviruses. PP319427 and PP319428 are genetically close to oral-associated Vientoviruses, and PP319424 is related to lung-associated Vientoviruses. Blue circles represent bootstraps ranging from 4 (smallest) to 100 (largest)
The prevalence of ReDoVs in nasopharyngeal samples
A set of primers was designed to encompass a conserved region from nucleotides 1709 to 1895 (amplicon size of 187nt), covering 54 nt end of the C-terminal region of the Capsid coding region and the gap before starting the Rep coding sequence. Figure 2 displays the PCR amplification and gel electrophoresis results, showing specific bands corresponding to the amplified ReDoV DNA. Additionally, the efficiency of the PCR was 99.3% (Fig. 3).
PCR amplification and gel electrophoresis of ReDoV genome. The gel electrophoresis demonstrates the presence of ReDoV DNA in the positive samples (Black arrows) with a size of 187 nt long. L100 represents the DNA marker (ladder) for size reference, while lane numbers show the PCR products of individual samples
Calculated PCR efficiency with a rounded slope of 3.34. 350 ng viral DNA was also detected by this PCR method
ReDoV positivity was detected in 190/731 (25.99%) cases, with 29/190 (15.26%) exhibiting coinfection with SARS-CoV-2. The contingency table analysis was used to examine the relationship between ReDoVs presence and SARS-CoV-2 infection. The chi-square test of independence revealed no association between ReDoVs and SARS-CoV-2 infections (p > 0.05). Similarly, Fisher’s exact test showed no significant association between ReDoVs and SARS-CoV-2 (p > 0.05). These results indicate no statistical link between ReDoVs and SARS-CoV-2 infection among the studied individuals.
Furthermore, the relationship between ReDoV infection and sex among individuals was investigated (Fig. 4). The positive results for ReDoVs among females and males were 135/731 (18.47%) and 55/731 (7.52%), respectively. No significant association was observed (p = 0.12), suggesting the variables’ independence. Furthermore, Fisher’s exact test for significance by cell also displayed no significant association between ReDoVs results and sex (p > 0.05).
Association of ReDoVs genome prevalence in different age categories, sex, SARS-CoV-2 results, and gum or dental complications results. The Red arrows highlight the groups in which negative or positive results of ReDoV are significant based on Fisher’s exact test
Additionally, the relationship between ReDoVs and age categories was evaluated. Accordingly, the results showed that among those aged 1–9, 10–19, 20–29, 30–39, 40–49, 50–65, and > 65, there were 0, 6, 40, 59, 42, 26, and 17 individuals positive for ReDoVs within the corresponding age categories. The chi-square test of independence (p > 0.05) indicated that infection and age categories at ReDoVs are independent. Further analysis using Fisher’s exact test for significance by cell revealed no significant association between ReDoVs genome presence and age categories. However, Fisher’s exact test showed a non-random association in the absence of the ReDoVs genome in children aged 1–9. Nevertheless, due to the limited number of patients in this category, the virus’s prevalence in this age group needs further investigation. No significant associations were also found between other clinical variables.
The association of ReDoVs with gum’s problems
The participants were questioned about any dental or gum inflammations or issues they had during their referral to the medical center. Surprisingly, the results revealed a highly significant relationship between infection with ReDoVs and dental/gum complications. The frequencies of negative and positive ReDoVs in the absence or presence of gum problems showed 126 individuals with gum problems tested positive for ReDoVs (p p 2.