The cohorts included in the study were from 5 countries on 4 continents. The study and all sampling were conducted in accordance with relevant guidelines and regulations.

For the veterinary cohort from Finland, we obtained serum samples from 324 healthy adult volunteers (Table 1). Samples were collected from participants at the national Annual Veterinary Congress in 2009 in Helsinki, Finland (25). Most (82%) of the volunteers were veterinarians, veterinary students, or veterinary nurses, and 92% completed an electronic questionnaire to obtain background information. Written informed consent was obtained from all study participants, and the study was approved by the Ethics Committee of Helsinki University Central Hospital.

For the cohort from the United States, we obtained serum samples from 84 healthy blood donors at Blood Systems Research Institute (San Francisco, CA, USA) (Table 1). The samples were collected during April 2009 in 2 locations (Arizona [n = 40] and Mississippi [n = 44]). Under US human and health service regulations, the study of preexisting, deidentified samples is not classified as human subject research.

For the cohort from Iraq, we obtained serum samples from 99 healthy adults (Table 1) to assess exposure of the population to various virus infections in Nasiriyah, Dhi Qar, in southern Iraq (26). Written informed consent was obtained from all study participants, and the study was approved by the Ethics Committees of Medical Sciences at Basrah University and the Al-Hussein Teaching Hospital.

For the cohort from Iran, we obtained serum samples from 107 healthy adults (Table 1) at the Hamadan Blood Transfusion Organization (Hamadan, Iran). Informed consent was waived for analysis of these deidentified blood donor samples, and the study was approved by the Ethics Committee of Hamadan University of Medical Sciences.

For the cohort from Kenya, we obtained serum samples from 107 children and 119 adults who had a febrile illness of unknown cause and had visited health clinics in Mwatate, Voi, or Wundanyi in Taita Taveta County in southern Kenya (Table 1). A questionnaire to obtain background information and symptoms was completed by all patients, and written informed consent was obtained from all study participants or guardians of children. The study was approved by the Kenyatta National Hospital–University of Nairobi Ethics and Research Committee.

We performed statistical analysis by using 2 × 2 tables and test statistics (mid p-exact value) in OpenEpi software (https://www.OpenEpi.com). A 2-tailed p value <0.05 was considered statistically significant.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis identified cutavirus VP2s of expected size (≈64 kDa) (Figure 1, panel A). Electron microscopy identified parvovirus-like VLPs with a diameter of ≈25 nm (Figure 1, panel B).

In the cutavirus IgG EIA, it was evident that bufavirus 2 and cutavirus cross-react: in all samples with an OD >0.5 for bufavirus 2 or cutavirus, a reaction was always observed with the other antigen. In samples showing weak reactivity (OD 0.1–0.5) in cutavirus or bufavirus 2 IgG EIAs, both cross-reactivities and single-specific reactivities were observed. In the competition assay, the specific reactivity of bufavirus or cutavirus was blocked completely only by homologous antigen, whereas cross-reactive reactions were blocked by homologous and heterologous antigens. In several instances, heterologous antigen slightly reduced the specific EIA reactivity; however, this reduction was much less than that caused by homologous antigen.

In cohorts from the Middle East and Africa that showed high prevalences of bufavirus IgG, some cross-reactivity was also observed among the 3 bufavirus genotypes, mostly for samples with higher ODs. However, the competition assay used for all positive samples could distinguish genotype-specific reactivity for correct interpretation. Tusavirus IgG did not cross-react with bufavirus IgGs or cutavirus IgG.

Bufavirus IgG was rare among veterinarians from Finland and blood donors from the United States: only 1.9% of the veterinarians and 3.6% of the blood donors had bufavirus IgG (Table 2). In these cohorts, each bufavirus IgG–positive person had antibodies against only 1 bufavirus genotype. No indications of specific animal contact being associated with bufavirus seropositivity were found when we compared background information for bufavirus IgG–positive and bufavirus IgG–negative veterinarians. For blood donors from the United States, all 3 bufavirus IgG–positive samples were from Mississippi. However, all samples from Arizona were negative for bufavirus IgG. The most commonly detected genotype was bufavirus 1 in Finland and bufavirus 3 in the United States (Table 2).

In striking contrast to adults from the United States and Finland, including our previous results for students and staff members from Finland (12), bufavirus IgG was common in Iraq, Iran, and Kenya, for which 84.8%, 56.1%, and 72.3%, respectively, of adult populations had IgG against >1 bufavirus genotypes (Table 2). In the Middle East, bufavirus 1 was the most common type, whereas in Kenya, bufavirus 3 was the predominant genotype (Table 2). Bufavirus 2 was the second most prevalent bufavirus in all 3 high-prevalence countries. In Iraq, we found that 30 (30.3%) of 99 persons had antibodies against 2 bufavirus genotypes, and 7 (7.0%) of 99 persons had antibodies against all 3 bufavirus genotypes. In adults from Kenya, we found similar prevalences: 30 (25.2%) of 119 persons had antibodies against 2 bufavirus genotypes, and 6 (5.0%) of 119 persons had antibodies against all 3 bufavirus genotypes. However, in Iran, we found that double or triple prevalences were lower: 14 (13.1%) of 107 persons had antibodies against 2 bufavirus genotypes, and 2 (1.9%) of 107 persons had antibodies against all 3 bufavirus genotypes.

In Kenya, HIV-positive patients had a similar bufavirus IgG prevalence as the rest of the cohort: 78.9% (30/38) in HIV-positive adults (mean age 46.3 years, range 27–85 years) vs. 69.1% (56/81) in HIV-negative adults (mean age 41.9 years, range 18–88 years) (p = 0.275). When we compared only the 38 HIV-positive persons and 60 HIV-negative persons within the same age range (27–85 years), bufavirus seroprevalences were even more similar: 79% for HIV-positive persons and 75% for HIV-negative persons (p = 0.669). However, possible undiagnosed cases of infection with HIV and unequal numbers could affect the accuracy of this comparison.

When the adult cohorts were analyzed more closely and persons were divided by age into equal-sized groups of <40 years of age and >40 years of age, we found that for younger adults in Iran, bufavirus seroprevalence was lower than that for older adults (21/54 [38.9%] vs. 39/53 [73.6%]; p = 0.0003). However, a similar distinction was not observed for adults from Iraq or Kenya (Table 3). For veterinarians from Finland and adults from the United States, we found that younger adults also had a lower bufavirus seroprevalence, albeit without statistical power, because of the low overall prevalence of bufavirus IgG in these countries. When we divided the cohorts into persons <30 years of age and >30 years of age, a similar trend was also observed for adults in Kenya (14/35 [56.0%] vs. 70/92 [76.1%]; p = 0.0595). This trend was not observed for persons in Iraq.

In Kenya, the bufavirus IgG prevalence in children was significantly lower than that in adults (20.6% in children <18 years of age vs. 72.3% in adults; p<0.0001), but we observed similar proportions of bufavirus genotypes and a predominance of bufavirus 3 in both adults and children. (Table 2). When we divided the cohort of children into those <5 years of age and those 5–17 years of age, the prevalence of bufavirus IgG by age increased from 12% to 28.1% (Figure 2).

The prevalence of cutavirus IgG was generally low for all groups, ranging from 1.0% in Iraq to 5.6% in Iran, and cutavirus IgG was not detected in adults in the United States (Table 2). In veterinarians in Finland, cutavirus IgG (4.9%) was more common than bufavirus IgG (1.9%) (p = 0.032). Two adults from Kenya and 1 veterinarian from Finland had both cutavirus IgG and bufavirus 2 IgG in their samples, which showed that these 2 antigenically similar viruses can infect the same person and elicit specific immune responses against each virus. However, for 6 patients (1 in Finland, 2 in Iraq, and 3 in Kenya), we could not determine whether the reactivity detected was specific for bufavirus 2, cutavirus, or both. These results were not included in the final prevalence calculations (Table 2). Tusavirus IgG was not detected in any cohort (Table 2).