Increased prevalence of anellovirus in pediatric patients with fever

Abstract

The Anelloviridae family consists of non-enveloped, circular, single-stranded DNA viruses. Three genera of anellovirus are known to infect humans, named TTV, TTMDV, and TTMV. Although anelloviruses were initially thought to cause non-A-G viral hepatitis, continued research has shown no definitive associations between anellovirus and human disease to date. Using high-throughput sequencing, we investigated the association between anelloviruses and fever in pediatric patients 2-36 months of age. We determined that although anelloviruses were present in a large number of specimens from both febrile and afebrile patients, they were more prevalent in the plasma and nasopharyngeal (NP) specimens of febrile patients compared to afebrile controls. Using PCR to detect each of the three species of anellovirus that infect humans, we found that anellovirus species TTV and TTMDV were more prevalent in the plasma and NP specimens of febrile patients compared to afebrile controls. This was not the case for species TTMV which was found in similar percentages of febrile and afebrile patient specimens. Analysis of patient age showed that the percentage of plasma and NP specimens containing anellovirus increased with age until patients were 19-24 months of age, after which the percentage of anellovirus positive patient specimens dropped. This trend was striking for TTV and TTMDV and very modest for TTMV in both plasma and NP specimens. Finally, as the temperature of febrile patients increased, so too did the frequency of TTV and TTMDV detection. Again, TTMV was equally present in both febrile and afebrile patient specimens. Taken together these data indicate that the human anellovirus species TTV and TTMDV are associated with fever in children, while the highly related human anellovirus TTMV has no association with fever.

Figure 1. Anellovirus DNA was more prevalent in specimens from febrile children by high-throughput sequencing.

A–B. High-throughput sequencing analysis of plasma (A) and NP (B) specimens showed that a higher percentage of specimens from febrile children contained anellovirus DNA compared to specimens from afebrile children (plasma, p = 0.034; NP, p = 0.002). C. The trend was the same, but not statistically significant at the patient level of analysis. D–E. Afebrile and febrile patients had a similar median number of anellovirus sequence reads in their plasma (10.2 vs. 15.6) (D) and NP (2.9 vs. 1.7) (E) specimens. F. Anellovirus sequences detected by high-throughput sequencing were more prevalent in plasma compared to NP specimens (p<0.0001; Chi-squared test).

Figure 2. PCR analysis showed that DNA from human anellovirus species TTV and TTMDV, but not TTMV, were more prevalent in febrile patient specimens compared to afebrile controls.

A–C. PCR identified TTV (A) and TTMDV (B) in a higher percentage of febrile patients compared to afebrile controls while TTMV (C) was present in equivalent percentages (plasma TTV, p = 0.0567; NP TTV, p = 0.0182; patient TTV, p = 0.0026). D. The majority of patients were either positive or negative for TTV, TTMDV, and TTMV DNA in both their plasma and NP specimens. P-values determined by chi-squared test.

Figure 3. Many patient specimens contained DNA from multiple human anellovirus species.

A–B. Patient plasma (A) and NP (B) specimens were assayed for TTV, TTMDV, and TTV DNA by PCR and the percentage of specimens with 3, 2, 1, or no anellovirus species were determined.

Figure 4. Anellovirus association with patient age, race, and gender.

A–F. Children in whom anellovirus DNA was detected were older than children in whom anellovirus DNAS was not detected. Patients were broken down by age into 6 month age groups and analyzed for the presence of DNA from TTV (A–B), TTMDV (C–D), or TTMV (E–F) in patient plasma (A, C, E) or NP (B, D, F) specimens. G. Other demographic analysis showed that anellovirus species TTV and TTMDV were more prevalent in patient specimens from African-Americans than Caucasians, but using multivariate logistic models the race effect was not significant after adjusting for the febrile status. H. There was no correlation between anellovirus positivity and patient gender.

Figure 5. Detection of anellovirus DNA correlates with increasingly elevated temperatures in children.

A–B. The percent of patients infected with detectable TTV (A) and TTMDV (B) rises as patient's temperature increases. C. The percentage of patients with TTMV DNA detected is independent of temperature.