The authors have declared that no competing interests exist.
Conceived and designed the experiments: IMVCV ACRV RI. Performed the experiments: IMVCV ACRV LFAM. Analyzed the data: IMVCV ACRV VNA LFAM MOGI RI. Contributed reagents/materials/analysis tools: ACRV MOGI RI. Wrote the paper: IMVCV ACRV MOGI RI.
JCV is the etiologic agent of progressive multifocal leucoencephalopathy (PML), a progressive neurological disease of the central nervous system
JCV infects the human host and establishes a persistent replicative cycle (including latency) following the acute infection, which favours its dissemination within human populations and its carriage through generations. It rarely appears to produce recognizable disease. Seroepidemiologic studies have shown that 70% to 80% of the adult human population shows the presence of antibodies to JCV, usually in the absence of clinical symptoms
The ancestral JCV probably generated three phylogenetic groups, designated as types A, B and C, which further mutated into subtypes that were distributed across the Old World, infecting human populations groups who were already there or were introduced into the geographic areas
Population studies based on the detection of viral DNA in urine shows the presence of JCV ranging from 20% in Africa to 65% or more in Asia and in Asia-descendant population groups
The present study aimed to describe the occurrence of JCV, including their types and molecular subtypes among human populations in the Brazilian Amazon and their distribution according to their ethnic background.
Urine samples from three groups were examined: urban (341 individuals residing in Belem, Para), Amerindians (42 individuals from 10 native Indian tribes from the Arara do Laranjal, Assurini do Trocara, Aukre, Kendjam, Munduruku, Parakanã, Surui, Tembe, Urubu-Kaapor and Wai-Wai villages) and 63 Afro-descendants living in the region of Rio Trombetas.
Variables | Urban | Amerindians | Afro-descendants |
Total examined | 341 | 42 |
63 |
Males | 154 | 3 | 38 |
Females | 187 | 38 | 23 |
Age range (yo) | 1–96 | 14–58 | 1–69 |
One individual did not have sex registered;
Two individuals did not have sex registered.
The individuals were briefed about the project and those who accepted to take part were given an inform consent to sign. The present work was submitted and approved by the Ethics Committee of the University Hospital Joao de Barros Barreto and followed the Brazilian Guidelines and Regulatory Standards for Research Involving Human Subjects (Resolutions 196/1996 and 347/2005 of the Brazilian National Health Council).
Urine samples (50 mL) were collected in vial collectors and transported to the Virus Laboratory of the ICB/UFPA and stored at −20°C until assaying for JCV infection. The urine samples were centrifuged at 5,000 rpm for 15 minutes, the urinary sediment washed three times with sterile saline solution and the cell pellet used to DNA extraction following the protocol using the EZ-DNA kit, Gentra Systems, Inc., USA.
Polymerase Chain Reaction - PCR was performed for amplification of the VP1 gene (215 bp) and the genomic region IG (610 bp) of the JCV, using a thermocycler (Mastercycler Personal, Eppendorf, Germany). The amplified products were subjected to purification for subsequent sequencing analysis of the nucleotide bases.
The reactions for the amplification of both segments (215 and 610 bp), were performed in a volume of 50 µL containing 400 ng of DNA extracted, 200 µM of each dNTP, 20 pmol of each primer, 50 mM KCl, MgCl2 2.0 mM, Tris-HCl pH 8.3 10 mM and 0.5 U of Taq DNA polymerase. The pair of primers involved for segment 215 bp was: (JLP-15-F)
The products of amplification were visualized after electrophoresis in agarose gel with 2% in TAE 1× buffer containing ethidium bromide and the aid of a transilluminator with a source of ultra-violet light. Purification of the products followed the protocol of the QIAquick PCR Purification Kit (Qiagen, Inc., USA).
Sequencing and phylogenetic analysis - The amplified products of the IG region were used for the construction of the phylogenetic trees and subsequent relationships among the strains detected. Fourty-nine samples were sequenced according to the quality of the amplified product which was generated. The products with a reasonable quantity provided good sequences suitable for the phylogenetic analysis. The amplified fragments were submitted to a direct sequencing assay (both forward and reverse) according to the protocol of the ABI Prism Dye Terminator Cycle Sequencing Ready Kit (Applied Biosystems, US) and the products were loaded on the ABI Prism 310 DNA Sequencer (Applied Biosystems, US). The nucleotide sequences were used together with other JCV sequences available in the Genbank (HR5, C24704; HR13, C24711; AT-2, C24690; AT-4, C24692; SD-9, C24753; IT-8, AU078543; KO-2, AU078521; KO-3, AU078520; KO-5, AU078518; ES-3, AU078596; IT-5, C22883; G2, AU078596; IT-2, C22881; SP-7, AU078565; N5, C22891; UK-2, C22875; G4, AB074580; GK-3, AB048563; SP-1, C22878; UK-1, AB004499; SW-3, C22886; MR-7, C22873; N25, AU078585; G5, C22894; IT-3, C22882; AM-18, AU078447; SI-1, AU078574; SI-7, AU078568; GH-2, D43780; GH-4, D43782; GH-3, D43781; GH-1, D43779; KE-1, C22847; ET-3, C22844; ET-1, AB004469; IN-1, C22790; SU-5, AB127019; GR17, AU078479; UZ-24, AB061161; NG-2, AB127004; SO-1, C22841; CW-2, C22715; ML-6, AB048581; ID-1, C22796; MO-11, C22761; AT-8, C24696; MY, AU062350; Tokyo-1, AF030085; TKY-1, AB038254; HR-7, C24706; MU-3, C22865; IN-6, C22794; MU-9, C22868; MO-2, C22755; MO-5, C22758; MO-3, C22756; SL-2, C22814; CB-3, C22747; MO-6, C22759; MO-1, AB048561; CY, JQ237149; ML-1, C22805; CB-2, C22746; C2, C22721; JS-K, CO897282; GS-B, AF004350; N4, C22890; SA-5, C22818 and SA-3, C22817. The sequences were alligned using the Phylip 3.56 software package
The geographical distribution of native Indian tribes and the African-descendants of original “quilombos” from the State of Pará (Trombetas) in relation to the city of Belém is depicted in
Tree was constructed using the Neighbor-Joining method after alignment of 440 nucleotides of the genomic region IG. Statistical support was performed by using 1,000 bootstrap.
Additionally, the molecular analysis showed the amplification of a nucleotide sequence of 579 bp, characteristic of BKV poliomavírus, showing 98.9% of identity with the PittVR4 line in one sample of the Trombetas “quilombo”.
Phylogenetic trees (Neighbor-Joining) constructed using the genomic sequences of the IG region (
Type B was mostly of Af2 subtype. Four samples of type B were classified as subtype MY (Asia), with 99% of bootstrap. These samples showed 99% similarity with the prototype MY (ME-4/MYd) described by Zheng et al.
The samples grouped as Type C (Type 6) belonged to subtype Af1 (African). This group presented the bootstrap value of 93%. A sample of the urban population of Belem was characterized as belonging to Type A subtype EU (bootstrap of 97%). The African-descendant samples of the Trombetas, elicited JCV strains Type B, subtypes MY and Af2. The analysis of a single sample of an indigenous community (tribe Surui), showed the presence of Type B subtype MY, also equivalent to the prototype MY (ME-4/MYd) described by Zheng et al.
The analysis of the sequences (data not shown) enabled the identification of specific mutations among viral subtypes. The samples belonging to type B, subtype MY presented mutations in specific positions: 2242 nt (C→A/G), 2285 nt (C→G), 2309 nt (A→G), 2362 nt (A→G) and 2407 nt (C→T). The samples grouped as Type C showed a T→G mutation at position 2353 nt. Moreover, the samples grouped in Type B, subtypes MY and Af2 showed mutations which were common to these subtypes at positions 2312 nt (G→A) and 2377 nt (T→C).
The present investigation is probably the first one among apparently healthy individuals in Brazil, as well as, among native Indians and Afro-descendant groups (originated from escapee slaves during the XIX century) in the North region of the Brazilian Amazon region.
The sex composition was balanced and followed the demographics of the city of Belem, the largest and most important city of the North region of the country
Among the Afro-descendants and the residents in Belem, older than 30 years of age, 65% and 66%, respectively, were infected, similarly to what has been described when testing new diagnostics kits, in Australia and among persons with non-Hodgkin lymphoma
JCV infection was defined by the detection of two genetic codifying areas (VP1 and IG regions), which present higher heterogeneity as compared to the regulatory region. The use of molecular biology assays is in agreement with seroepidemiological studies to determine the prevalence of the virus
In the present paper it was possible to amplify both regions in 77%, 52% and 16% of samples from Amerindians, urban groups and Afro-descendants, respectively. The efficiency of amplification of IG is highly dependent on the supercoiling nature of polyomaviruses
The prevalence of JCV among residents in Belem (33%) is comparable to those found in Australia (29%)
The presence of JCV in one Surui Indian woman is a good indicator that the virus may be circulating among the community as they still are an epidemiologically semi-closed population. The small number of individuals examined may have accounted for the sole finding. A large variation of prevalence rates has been reported in the Americas that includes the Inuits in Canada (26%)
JCV infections among Afro-descendants is usually similar. The prevalence among pigmies was 22% and 20% among Bantus
JCV is usually associated to population groups and their migration patterns in the same fashion as with mitochondrial DNA and the Y chromosome
In Belem, three ancestral types were described, according to its geographical association to the type A (3%), found in Europe, type B (83%), found in Africa and Asia, and type C (14%) found in the African continent. This is in strict adherence to the historical process of formation of the population of urban communities like Belem, as well as to the genetic information available of the composition of the urban population of Belem using classic genetic polymorphisms
Type B was the most frequently found and samples from Belem which were subtyped as Af2 (66%) were mostly from “mesticos” (84%) who were born in the city as well (56%). It is relevant to mention that 25% of the individuals presented in their demographic records the observation of phenotype characteristics related to a black ethnicity, but without a self referral as such. This is a relevant matter when considering that most of the Af2 genotype evolved within the African continent approximately 50,000 years ago
All samples from Belem classified as subtype MY were originated also from individuals classified as “mesticos”. Two were born in Belem and two were born in two different villages. The samples showed 99% similarity to the MY prototype described among native Americans
The high similarity observed among the samples from urban areas, the native Indians and the prototype isolated from native Americans is suggestive that type B, subtype MY, entered into the Brazilian Amazonian population during its process of formation by the mixture of migration patterns more than 10,000 years associated to the introduction of the European and the incoming African groups.
JCV is shown herein, for the first time, as a strong marker of population migration patterns when the similarity of the American strains are compared, including those of epidemiologically closed communities originated after the crossing of the Behring Strait and settling in the Americas
In a similar way, the strain was introduced in the city of Belem probably from its origin in 1616 due to the extensive mixture of the three ethnical groups. The contribution of the Indian population within urban areas is approximately 35% and to a lower extent, that of the Afro-descendants
The phylogenetic relationship among JCV strains from Japan, Korea and Amerindians suggests that emergence of subtype MY occurred between 50 and 30 thousand years ago and that within the MY clade between 30 and 10 thousand years
The role of JCV as a marker of past and recent human migration and mixture of different ethnic groups is also defined by the detection of type C, in which all samples were identified as subtype Af1. The isolates were originated from individuals with phenotypic and socio demographic informations of being all “mesticos” of whom 60% resembled whites and 40% negros. Most of them were born outside large cities.
Type C represents the ancestral JCV that evolved circa 100 thousand years ago, generating subtype Af1, which was subsequently disseminated into West Africa, an area associated to the emergence of the human species, and later to Central Africa including Central African Republic, Ghana and Mauritania
It has been proposed that the ancestral JCV emerged together with man (circa 200–100 thousand years ago) in Africa
The first modification that took place in type B, originated Af2 and the non-Af2 subtypes. Subtype Af2 was then disseminated within the African continent and, secondarily, to Saudi Arabia, the West and South of Asia, as well as to Southeast Europe (circa 50 to 10 thousand years ago). The non-Af2 strains originated the B1-c, a minor European genotype, and the main genotypes present in Asia, Oceania and the Amerindians from North America (B1-a, B1-b, B1-d, B2, CY, MY, SC, 8A, 8B e 2E).
Type A, subtype EU was also detected within the urban population of Belem, from one individual whose relatives dated back to the routes of heavy migration from Portugal during colonization. This recent migration pattern of Europeans, particularly in the last 300 years, enables the role of JCV as a good marker to trace patterns of ancient and recent migration of humans and its genetic mixture with other ethnic groups.
Mutations in positions 2242 nt (C→A/G), 2285 nt (C→G), 2309 nt (A→G), 2362 nt (A→G) and 2407 nt (C→T), may be considered as a signature of the viral subtype after entering the Amazon region of Brazil. Maintenance of JCV is a consequence of persistence in the human host, its moderate genetical stability and the low levels of DNA mutations. The presence of the same pattern of mutations even within a woman from the Surui native Indians, a semi-closed epidemiological community, reinforces the genetic peculiarity of subtype MY in the Amazon region of Brazil and the transmission of the virus from the ancestral human native populations to the Afro-descendants and the present tri-hybrid human populations.
It was unexpected the finding of a nucleotidic sequence of the polyoma virus BK in an Afro-descendant individual, but it was previously described among patients who received bone marrow transplants
We thank the subjects from Belem, the Indian tribes and the Trombetas and send them our best wishes.