R n tr t n P p l t n H t r n J V r : r nd ,p n h, nd fr n n th n tr f d rn P rtR nMariana Fernandez Cobo, David V. Jobes, Richard Yanagihara, Vivek R. Nerurkar,

Yasuhiro Yamamura, Caroline F. Ryschkewitsch, Gerald L. Stoner

Human Biology, Volume 73, Number 3, June 2001, pp. 385-402 (Article)

P bl h d b n t t n v r t PrDOI: 10.1353/hub.2001.0032

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Reconstructing Population History Using JC Virus: Amerinds,Spanish, and Africans in the Ancestry of Modern Puerto Ricans

MARIANA FERNANDEZ COBO,1,4 DAVID V. JOBES,1,5 RICHARD YANAGIHARA,2

VIVEK R. NERURKAR,2 YASUHIRO YAMAMURA,3 CAROLINE F. RYSCHKEWITSCH,1

AND GERALD L. STONER1

Abstract The roots of the Hispanic populations of the Caribbean Islandsand Central and South America go back to three continents of the Old World.In Puerto Rico major genetic contributions have come from (1) Asians in theform of the aboriginal Taino population, an Arawak tribe, present whenColumbus arrived on the Island, (2) Europeans, largely Spanish explorers, set-tlers, government administrators, and soldiers, and (3) Africans who came aspart of the slave trade. Since JC virus (JCV) genotypes characteristic of Asia,Europe, and Africa have been identified, and excretion of JCV in urine hasbeen proposed as a marker for human migrations, we sought to characterizethe JCV strains present in a Caribbean Hispanic population. We found that thestrains of JCV present today in Puerto Rico are those derived from the OldWorld populations represented there: Types 1B and 4 from Spain, Types 3A,3B, and 6 from Africa, and Type 2A from Asia. The Type 2A genotype repre-sents the indigenous Taino people. This JCV genotype was represented muchmore frequently (61%) than would be predicted by the trihybrid model of ge-netic admixture. This might be attributable to characteristics of JCV Type 2Aitself, as well as to the nature of the early relationships between Spanish menand native women. These findings indicate that the JCV strains carried by theTaino Indians can be found in today’s Puerto Rican population despite the ap-parent demise of these people more than two centuries ago. Therefore, molec-ular characterization of JCV provides a tool to supplement genetic techniquesfor reconstructing population histories including admixed populations.

The ancestry of Hispanic populations in the New World includes contributionsfrom Europeans (mainly Spanish), Africans brought by the slave trade (Rout

1Neurotoxicology Section, National Institute of Neurological Disorders and Stroke, National Institutes ofHealth, Bethesda, MD 20892.

2Retrovirology Research Laboratory, Pacific Biomedical Research Center, University of Hawaii at Manoa,Honolulu, HI 96822.

3Ponce School of Medicine, AIDS Research Program, Ponce, Puerto Rico 00732.4Servicio de Biologia Molecular, Departamento de Virus, ANLIS – INEI, (1281) Buenos Aires, Argentina.5Present address: Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404.

Human Biology, June 2001, v. 73, no. 3, pp. 385–402.Copyright © 2001 Wayne State University Press, Detroit, Michigan 48201-1309

KEY WORDS: POLYOMAVIRUS, JC VIRUS, GENOTYPES, HISPANIC AMERICANS, MIGRATION,GENETIC ADMIXTURE

386 / fernandez cobo et al.

1976), and the various Native American peoples inhabiting the lands colonized atthe time of contact (Morner 1967). This trihybrid genetic model of Hispanic pop-ulations in the New World is well attested both by history and by genetic studiesof blood groups and enzyme polymorphisms (Sans 2000). In Mexico, theCaribbean, as well as in Central and South America, the proportion of these threemajor strands in the national genetic fabric is different and varies from communi-ty to community (Table 1). In Puerto Rico and among Mexicans a major compo-nent of the modern gene pool is thought to have come from Spanish immigrants(Lisker et al. 1986; Hanis et al. 1991; Long et al. 1991; Lisker et al. 1996). TheAfrican contribution varies greatly and is higher in Puerto Rico than in Mexico,while the available evidence suggests that the Amerind contribution is lower inPuerto Rico (Hanis et al. 1991). More recently, methods for applying moleculargenetic data for analysis of admixed populations have been developed (Shriver1997; Bertorelle and Excoffier 1998).

The modern history of Puerto Rico began with the arrival of Columbus onNovember 19, 1493, at the island he named San Juan Bautista (called Boriquénby the Indians). The aboriginal inhabitants with whom Columbus made contactduring this second voyage were largely the peaceable Taino Indians, an Arawaktribe, rather than the more warlike Caribs (Rouse 1948; Rouse 1993). A perma-nent Spanish outpost was established in 1508. As elsewhere in the Caribbean, thenative population was soon greatly reduced by warfare, disease, and slavery(Rouse 1993). In 1511 an Indian rebellion against the crown was brutally sup-pressed (Alegria 1974). Many fled to the hills or left the island. The Spanishcrown gave permission for the importation of African slaves to the Americas, andAfricans were brought to Puerto Rico by early settlers and later slave traders tofill the demand for laborers. Eventually, African slaves replaced Tainos as labor-ers in the plantation system that developed.

Table 1. Genetic Admixture Percentage in Trihybrid Hispanic Populations

PopulationEuropean

(%)Amerindian

(%)African

(%) Reference

Puerto RicanSan Juan, PR 45 18 37 Hanis et al. 1991

MexicanMexico City 41 56 3 Lisker et al. 1986Starr Co., TX 61 31 8 Hanis et al. 1991Scottsdale, AZ 68 29 3 Long et al. 1991

CubanHavana, Cuba 62 18 20 Hanis et al. 1991

BrazilianNortheast Brazil 59 11 30 Krieger et al. 1965Manaus 61 27 12 Santos et al. 1983

JC Virus in Puerto Rico / 387

The early Spanish immigrants, including government officials and soldiers,were mostly men. Spanish men continued to arrive in large numbers, many seek-ing gold. These adventurers, as well as later plantation owners and operators,sought liaisons with native women. In 1514 Spanish men were given permissionby the crown to marry Taino women, and in Puerto Rico, as elsewhere in LatinAmerica, many solemnized their unions in marriage (Morner 1967; Alegria 1974;Rouse 1993). In the African group as well as in the Spanish population, the malesgreatly outnumbered females. In a census of African slaves in 1530, the ratio ofmen to women was more than 4:1 (Jimenez de Wagenheim 1998). In SouthAmerica, Africans who managed to escape slavery sought refuge in Indian vil-lages, and some of their descendants became tribal leaders (Rout 1976).

The native population of Puerto Rico is generally estimated at 30,000 to50,000 at the time of contact. In 1765 Lieutenant General Alexander O’Reillyconducted a census that put the number of free people at 39,846 with the numberof slaves at 5,037 (Morales Carrion 1983). Native Taino Indians would have beenincluded in both groups, but their numbers are uncertain. In a census taken in1787 the natives, who were then living in interior villages, were found to numberonly 2,302 (Alegria 1974). By the end of the century these were gone, possiblydue to migration or starvation and disease, as well as to assimilation by the largerpopulation.

Although the Tainos as a distinct people disappeared about 200 years ago,there had been significant admixture occurring for nearly three centuries. Recent-ly, analysis of blood group and protein markers using a trihybrid model deter-mined that the Native American contribution to the Puerto Rican gene pool wasabout 18% (Hanis et al. 1991). The estimated European and African contributionswere 45% and 37%, respectively (Table 1).

JC virus (JCV), a human polyomavirus distributed ubiquitously, was firstrecognized as the viral agent associated with progressive multifocal leukoen-cephalopathy (PML) (Padgett et al. 1971). It has been found to occur in distinctgenotypes and subtypes representing the major continental dispersals of the hu-man population (Sugimoto et al. 1997; Agostini et al. 1997b; Chima et al. 1998).These viral genotypes are detectable in the urine of 20%–80% of adults by thepolymerase chain reaction (PCR) (Kitamura et al. 1990; Agostini et al. 1996;Shah et al. 1997; Agostini et al. 1997b). A European genotype of JCV, Type 1(Agostini et al. 1998a) (also known as Type A [Guo et al. 1996]) with two sub-types has been identified in Northern and Central Europe. Recent studies havealso identified Type 1 strains in Spain (Bofill-Mas et al. 2000). A closely relatedgenotype, Type 4, was originally described in the USA, but is also found in partsof Europe (H.T. Agostini, personal communication). In Africa Type 3 with twosubtypes and Type 6 have been recognized (Guo et al. 1996; Agostini et al. 1997a;Chima et al. 1998). Type 3A is present in East, Central, and West Africa; whileType 3B and Type 6 have been found only in West and Central Africa (Agostini etal. 1997a; Chima et al. 1998). Several Asian types and subtypes have been de-scribed. The major Asian genotypes are Type 2A in Northeast Asia (Guo et al.

388 / fernandez cobo et al.

1996; Guo et al. 1998; Agostini et al. 1998d) and Type 7 in South China andSoutheast Asia (Chang et al. 1996; Ou et al. 1997; Jobes et al. 1998). These can becompared directly to sequences designated as MY and SC by Yogo and cowork-ers (Sugimoto et al. 1997). To date, the large majority of JCV strains identified inNative Americans, i.e., the Navaho and Flathead people, were the Type 2A strainscharacteristic of Northeast Asia (Agostini et al. 1997a). No strains representativeof Southeast Asia (Type 7) have been found in the Americas. In the studies re-ported here we have determined the JCV genotypes excreted by Puerto Ricanadults. We provide evidence that the three major contributions to the contempo-rary Puerto Rican Hispanic gene pool (Native American, European, African) areeach represented by appropriate JCV genotypes in the adult population.

Materials and Methods

Study Population. We tested 55 healthy volunteers from Puerto Rico. Twen-ty-nine urine samples were collected anonymously from adults in a retirementhome in Ponce, which is centrally located on the southern coast about 50 milessouthwest of San Juan. All were Hispanics who had lived most of their lives inPuerto Rico. An additional 26 urine specimens were collected from Hispanicadults attending an international conference in San Juan. These were 19 women,5 men, and 2 of unrecorded gender. The age range was 23–47 yr for women and36–55 yr for men. Of the 55 study participants, 52 were born in Puerto Rico ofPuerto Rican parents and grandparents. Of the remaining three, one each wasborn in Cuba of Cuban parents, in Colombia of Colombian parents, and in El Sal-vador of El Salvadoran parents.

Specimen Handling. Urine specimens were stored at 4oC and shipped to theNational Institutes of Health in Bethesda, Maryland, for processing and analysis.Urine (10–15 mL) was centrifuged at 2000×g for 10 min. The cell pellet was re-suspended in 10 mL phosphate-buffered saline, re-centrifuged, and the super-natant discarded. Viral DNA was then extracted from the cell pellet using the QI-AampViral RNA/DNA kit (Qiagen) according to the manufacturer’s protocol.

Polymerase Chain Reaction. For detection of JCV-positive specimens andpreliminary viral genotyping, a 215-base-pair (bp) fragment was amplified fromthe VP1 major capsid protein gene using the previously described primer pair(Agostini et al. 1997b): JLP-15, nucleotides 1710–1734, 5′-ACAGTGTGGCCA-GAATTCACTACC-3′ and JLP-16, nucleotides 1924–1902, 5′-TAAAGCCTCC-CCCCCAACAGAAA-3′). This fragment provides sites that identify at least sev-en genotypes and additional subtypes and has been validated by the analysis ofcomplete JCV genomes (Jobes et al. 1998; Agostini et al. 1999). Polymerasechain reaction (PCR) conditions have been described (Ryschkewitsch et al.

JC Virus in Puerto Rico / 389

2000). PCR products were observed by separation on a 2% agarose gel (0.5µg/mL ethidium bromide) and visualized with ultraviolet light. Products were pu-rified on a QIAquick PCR purification (Qiagen) column and sequenced for posi-tive identification by manual cycle sequencing.

In samples from which JCV DNA was amplified using primers JLP-15 and-16, the viral regulatory region was amplified using primers JRR-25 and -28(JRR-25, nucleotides 4981–5003, 5′-CATGGATTCCTCCCTATTCAGCA-3′;JRR-28, nucleotides 313–291, 5′-TCACAGAAGCCTTACGTGACAGC-3′),which generate a 463-bp fragment encompassing the entire control region on bothsides of ori. This amplified fragment includes three typing sites to the early sideof ori (Agostini et al. 1997b). Regulatory region products of proper length wereexcised, purified with a QIAquick gel extraction kit (Qiagen), and sequenced bymanual cycle sequencing.

Manual Sequencing. Amplified fragments were sequenced with the ThermoSequenase Radiolabeled Terminator Cycle Sequencing Kit using 33P ddNTPs(USB Corp., Cleveland, OH) with the primers used for amplification. Productswere analyzed on Castaway 6% polyacrylamide gels (Stratagene, La Jolla, CA),then fixed with 5% methanol/5% acetic acid. After drying in a Castaway gel dry-er, the gel was exposed to Kodak BioMax MR X-ray film for 16–48 hr.

Complete-Genome Amplification. Complete-genome JCV DNA amplifica-tion utilized the primers BAM-1 and -2 as described (Agostini and Stoner 1995).Following amplification with the proofreading enzyme rTth DNA polymerase XL(Applied Biosystems), PCR products were separated on a 1% agarose gel contain-ing 0.5 µg/mL ethidium bromide and visualized under ultraviolet light. The band at5.1 kilobase pairs (kb) was excised and purified using the QIAquick gel extractionkit (Qiagen) (Jobes et al. 1999). For some virus strains (#241A, 242A, 313B, and604) the whole genome amplification was repeated six times to obtain sufficientviral DNA for automated sequencing using the ABI Prism BigDye Terminator Cy-cle Sequencing Kit and an ABI 373A Automated DNA Sequencer (AppliedBiosystems). Another strain (#603) was cloned and sequenced as described below.

Complete-Genome Cloning and Sequencing. PCR products generated usingthe rTth DNA polymerase were modified prior to TA cloning to contain singleadenine (“A”) overhangs, as described in Jobes et al. (1999). Three microliters (3µL) of PCR products were added to a solution containing 2.0 µL 10X PCR bufferwith 1.5 mM MgCl2, 0.8 µL 10 mM dNTP, 0.5 µL Taq DNA polymerase (2.5 U),and 13.7 µL ddH2O and incubated at 72°C for 15 min and then placed on ice.

The “A” modified PCR products were cloned using the TOPO-TA cloningkit (Invitrogen) and plasmid DNA was isolated and sequenced as described in(Jobes et al. 1999). Briefly, 2.0 µL of PCR product were used for the TA cloningfollowing the manufacturer’s recommendations. The ligation reaction was al-

390 / fernandez cobo et al.

lowed to proceed at room temperature for 5 min, then immediately placed on ice.The ligation products were used to transform TOP10 F′ competent cells. Fifty and100 µL of the transformation were plated onto separate LB agar plates with 100µg/mL ampicillin and incubated overnight at 37°C.

Multiple white colonies were chosen from the agar plates and grown in LBbroth (100 µg/mL ampicillin) overnight at 37°C with vigorous shaking (225rpm). Plasmid DNA was purified from 3.0 mL cultures using the QiagenMiniprep Kit and digested with BamHI to detect the presence of inserts. Equimo-lar amounts of plasmid DNA were pooled into a single tube and sequenced on anABI 373A automated DNA sequencer using the ABI Prism BigDye TerminatorCycle Sequencing kit (Applied Biosystems).

Genotyping. Genotypes were assigned in the JCV VP1 gene typing region bycomparison to prototype sequences of the known genotypes by inspection (Agos-tini et al. 1995; Agostini et al. 1996; Agostini et al. 1997b; Jobes et al. 1998;Agostini et al. 1998c). DNA sequence numbering of the coding region is that ofFrisque et al. (1984), and the archetypal control region is that of Yogo et al.(1990).

Phylogenetic Analysis of Complete JCV Genomes. The assignment of com-plete JCV genomes representing the Type 2A genotype (#241A and #242A), theType 3B genotype (#313B), and two Type 6 strains (#603 and #604) was exploredas follows. These genomes and 21 complete JCV genome sequences describedpreviously (Jobes et al. 1998) were aligned using the PileUp program of the Ge-netics Computer Group (GCG) Wisconsin Package, version 10.0-UNIX (Madi-son, WI). The gap creation penalty was set to 5 and the gap extension penalty to1. The Type 5 isolate (#501) from California was omitted from this analysis dueto its possible origin as a recombinant strain. For the phylogenetic analysis theregulatory region rearranging in PML brain (nucleotides 1–267) was omitted. Theresulting data was comprised of 4856 total characters (with gaps) and gaps weretreated as missing data. A single distance tree was then constructed using theheuristic search with the minimum-evolution option in the PAUPSearch programof the Wisconsin Package and was rooted using PAUPDisplay with the midpointmethod (part of PAUP 4.0) (Swofford 2001). Additional analysis utilized the SE-QUENCHER (Gene Codes Corp.) and DNASIS (Hitachi Software) programs ona desktop personal computer.

Reference Sequences. For the GenBank accession numbers of previouslypublished strains used in construction of the phylogenetic tree see Jobes et al.(1998). The new Puerto Rican Type 2A strains reported here have GenBank ac-cession numbers as follows: #241A and #242A have accession numbersAF295733 and AF295734, respectively. The Puerto Rican Type 3B strain

JC Virus in Puerto Rico / 391

(#313B) has accession number AF295735, and the Puerto Rican strains of Type 6(#603 and #604) have accession numbers AF281614 and AF295736, respectively.

Results

Seropositivity for JCV in adults ranges from 70%–90%, and many infectedbut asymptomatic individuals excrete JCV chronically or intermittently in theurine (Agostini et al. 1996; Guo et al. 1996). A sensitive primer pair for detectionof JCV DNA in urine amplifies a segment near the 5′ end of the VP1 gene that isalso the most informative region for genotyping. Of the total of 55 urine samplesstudied, 31 (56%) were JCV positive when tested using primers JLP-15 and -16(Table 2). Additionally, the regulatory region was amplified from 18 of the 31JCV-positive samples.

Genotypes representative of all three continental groups were present.Types 1 and 4, which are closely related European types, were each 6.5% of thetotal, giving a combined value of 13% for the European contribution (Table 2).Type 1 was confined to Type 1B. Type 1A, found in the USA and central Europe(Hungary) (Stoner et al. 1998) was not observed. The African genotypes repre-sented included all those identified to date, namely, Type 3A, Type 3B, and Type6 (total 26%). The only Asian genotype found, Type 2A (61%), was overrepre-sented compared to the predicted Native American contribution in the gene pool(18%; see Table 1). The Cuban and Colombian urine specimens each showedType 2A strains, while the El Salvadoran specimen was negative for JCV. Type2A is a Northeast Asian genotype apparently contributed by the Taino Indians.The JCV Type 7 strains found in South China and Southeast Asia were not ob-served in our study population. The basis for assignment of genotypes based onthe partial VP1 gene sequence is illustrated in Table 3.

Table 2. JCV Genotype Distribution among 31 Strains from Puerto Rico

JCV Genotype Number % Region of Origina

1B 2 6.5 Europe (Spain)4 2 6.5 Europeb

2A 19 61.3 Asia (Native American)c

3A 4 12.9 Africa3B 2 6.5 W. Africa6 2 6.5 W. Africa

a. Likely Old World origin of these Puerto Rican strains.b. Type 4 is a European strain, present in a minority of the modern Spanish population (unpublished

data).c. Presumably derived from the Taino (Arawak) Indian inhabitants of Puerto Rico.

392 / fernandez cobo et al.

Tabl

e 3.

Poly

mor

phic

Site

s in

the

VP

1 G

ene

Typi

ng R

egio

n of

JC

Vfr

om P

uert

o R

icoa

Nuc

leot

ide

Pos

itio

n

JCV

Type

sSt

rain

s17

5617

7117

8617

9018

0418

0518

1818

4318

5018

6918

70

Type

1B

2A

CC

GT

TA

GT

GG

GTy

pe 4

2A

CC

GT

TA

CG

AC

ATy

pe 2

A19

AC

AT

TT

AC

TA

GA

Type

3A

4T

CA

AT

CA

CT

AC

ATy

pe 3

B2

TC

AG

TT

AC

TA

CG

Type

62

AC

AG

CT

AC

TA

GG

a.Si

tes

at w

hich

str

ains

dif

fer

from

the

maj

or S

pani

sh g

enot

ype

(Typ

e 1B

) ar

e in

bol

d ty

pe.

1753

No.

of

JC Virus in Puerto Rico / 393

Of the 18 regulatory regions amplified, 6 of 8 Type 2A strains containedmultiple deletions including 10 bp at nucleotide positions 53–62, 1 bp at position74, and 2 bp at positions 221–222 (Figure 1). Similar 10-bp deletions in this re-gion have been observed in individual strains from Africa (Agostini et al. 1995;Chima et al. 1998) and in a group of variants found in Papua New Guinea(Ryschkewitsch et al. 2000). The three Type 3A strains have C at position 133, asdo African Type 3 strains previously described (Agostini et al. 1995; Chima et al.1998). The Puerto Rican Type 3B strains, however, did not have C at position133, in agreement with previous findings in an African-American Type 3B strain(Agostini et al. 1997a). The typing sites to the early side of ori were consistent inall strains with the typing determined from the VP1 gene (Table 4).

The whole genomes of five Puerto Rican strains were amplified and se-quenced (#241A, #242A, #313B, and #604) or cloned and sequenced (#603). Ofthese, two represented Type 2A, one Type 3B, and two Type 6. These genomes,exclusive of the regulatory region to the late side of ori that is prone to rearrange-ment in PML brain, consisted of 4853 to 4855 base pairs, and were subjected tophylogenetic analysis by a minimum-evolution distance algorithm (Figure 2).The two Type 2A sequences (#241A and #242A), which differed from each otherby only 0.06%, were found to be closely related to other Type 2A sequences ob-tained from Native Americans in New Mexico (#225A, #226A, and #228C)(Agostini et al. 1997a) and to the prototype Type 2A strain from Japan, Tokyo-1.The putative Type 3B (#313B) sequence was nearly identical to the Type 3B se-quence in an African American (nucleotide differences at only two positions)(Agostini et al. 1997b). Other Type 2A strains include one from a California His-panic (#224A), and another Type 2 strain from a California Caucasian (#229C)(Jobes et al. 1998) (Figure 2). The new Type 6 sequences (#603 and #604) werefound to be most closely related to the Type 6 prototype whole genome sequenceobtained originally from the brain of an African American progressive multifocalleukoencephalopathy (PML) patient (Agostini et al. 1998c). These new Type 6strains, which differed from each other by 0.39%, each differed from the proto-type Type 6 strain (#601) by 0.60% and 0.52% of their sequence, respectively. Wehave operationally defined JCV genotypes as sequences that differ by about 1.0%or more, while subtypes differ by approximately 0.5% to 1.0%. Subtypes havenot been assigned to Type 6.

Discussion

In many countries of the Caribbean, as well as in Central and South Ameri-ca, the majority of the population has three major genetic components: Spanish(or Portuguese), African, and Amerind. These are found in varying proportions,and as the founders of each of these population stocks differ from region to re-gion, so each New World Hispanic population is different. We show here that inPuerto Rico the major genetic contributions can be correlated with the residualgenotypes of JC virus passed to the descendants of these founding populations.

394 / fernandez cobo et al.

Fig

ure

1.Sc

hem

atic

rep

rese

ntat

ion

of r

egul

ator

y re

gion

seq

uenc

es to

the

late

sid

e of

ori

. Poi

nt m

utat

ions

fro

m a

rche

type

CY

are

indi

cate

d ab

ove

the

line.

Oth

erw

ise

the

sequ

ence

mat

ches

arc

hety

pe. D

elet

ions

are

ind

icat

ed b

y ga

ps i

n th

e lin

e. T

he s

eque

nce

illus

trat

ed i

s th

e do

min

ant

one

for

each

geno

type

. For

a li

stin

g of

thes

e an

d ad

ditio

nal m

utat

ions

see

Tab

le 4

.

JC Virus in Puerto Rico / 395

Table 4. Regulatory Region Sequence Mutations on the Early and Late Side of ori in18 JCV Strains from Puerto Rico

JCVGenotype

No. of Strains Mutationsa Deleted Positions

Typing Siteson Early Side

of orib

1B 2 217-A — A,C,C4 2 217-A — A,C,C2A 6 159-A, 217-A 53–62, 74, 221–222 T,T,G″ 1 217-A — ″″ 1 5025-Ac — ″3A 2 133-C — T,T,C″ 1 108-A, 133-C ″3B 1 — — T,T,C″ 1 94-T 95 ″6 1 — — T,T,C

a. Mutations on the late side of ori relative to archetype CY (Yogo et al. 1991).b. Positions shown are nucleotides 5017, 5026, and 5039, respectively.c. Mutation from “G” in Mad1 on the early side of ori (Frisque et al. 1984).

From the dominant European population, namely, the Spanish, Types 1B andType 4 were contributed. The African immigrants brought Types 3A and 3B, andmainly from West and Central Africa, Type 6 (Agostini et al. 1995; Agostini et al.1997a; Chima et al. 1998). The native Taino Indians presumably contributed Type2A strains, the same genotype found to dominate in the Navaho and the Flatheadpeople (Agostini et al. 1997b). Thus, the profile of extant genotypes of JCV inPuerto Rico can be linked with the well-established history of the island popula-tion. These results parallel those showing that urban African Americans retain Af-rican strains of JCV (Type 3), while also harboring European strains consistentwith a history of genetic admixture (Chima et al. 2000).

While the genotypes of JCV present in Puerto Rico are those expected fromthe ancestry of the population, the proportion of these different types is surpris-ing. The number of Type 2A strains, presumably contributed by the long extinctTaino Indians, far exceeds that predicted by the trihybrid model based on analysisof polymorphic blood group and protein genetic markers. If Taino genes make uponly 18% of the current Puerto Rican gene pool as others have calculated (Haniset al. 1991), why should Type 2A strains make up 61% of the JCV strains foundin this study?

Where there is intermarriage between populations harboring different JCVgenotypes, as in Puerto Rico, these genotypes come into direct competition for in-fection of the offspring. At the most basic level it is likely that a genotype whichis excreted earlier in life, more frequently, and more heavily, would be more like-ly to be transmitted to the next generation and thus would have a selective sur-vival advantage. Our evidence from the Navaho and Flathead peoples indicates

396 / fernandez cobo et al.

Figure 2. Phylogenetic tree including five new Puerto Rican complete genomes. A distance treewas reconstructed with 26 JCV genomes using the minimum-evolution method in thePAUPSearch package of programs and was rooted with the midpoint method usingPAUPDisplay. Types 1 and 4 represent strains of European origin. Strains of African ori-gin are Types 3 or 6. Strains of Asian origin are Types 2A/C, 2B, 2D, or Type 7. NewPuerto Rican genomes are indicated by boldface type. The Puerto Rican Type 2A strains(#241A and 242A) are closely related to the other Type 2A strains, including ones fromJapan (Tokyo-1), Native Americans (#225A, #226A, #228C), and a Hispanic individualfrom California (#224A) (Jobes et al. 1998). The new Type 3B (#313B) and Type 6strains (#603 and #604) are most closely related to strains of those types defined origi-nally in African Americans and Africans (Agostini et al. 1997a; Agostini et al. 1998c).

JC Virus in Puerto Rico / 397

that they excrete JCV more frequently (65%–80%) (Agostini et al. 1997b) thando Europeans (40%) (Stoner et al. 1998) or Africans (20%) (Chima et al. 1998). Itmight also be predicted that they excrete virus earlier in life, although there is lit-tle experimental evidence to support this point. During five centuries of directcompetition between viral genotypes in Puerto Rico, it is probable that Type 2Astrains have had a slight selective advantage. Thus, the genotype distribution mayhave become skewed, and may now overrepresent the proportion of NativeAmerican ancestry in the gene pool of the population. Nevertheless, none of theother major viral genotypes known to represent the founding populations hasbeen eliminated from our study sample.

In addition to viral factors, cultural factors may have contributed to the ap-parent overrepresentation of Type 2A in our Puerto Rican cohort. First, many ofthe early liaisons on the island were between native women and Spanish men whowere soldiers or in other forms of government service and arrived without wives.It is likely that the children were in the care of the mother, and being more heavi-ly exposed to the mother than to the father, were more likely to acquire the moth-er’s JCV genotype (Type 2A) than the father’s (Type 1 or Type 4). This type ofdirectional or asymmetrical mating has been studied by maternal (mtDNA) andpaternal (Y chromosome) markers in trihybrid Hispanic populations (Merri-wether et al. 1997; Bortolini et al. 1999). The Amerind contribution is alwayshigher when maternal lineages are analyzed.

Finally, as noted above, the excretion rate of JCV varies in different popu-lations of the world. Whether this is due more to the influence of human or viralgenetics is not yet clear. Nevertheless, the number of excreters may not accurate-ly reflect the actual number of infections by each genotype (assuming that nearlyall individuals are latently infected). For this reason, a part of the skewing towardType 2A may be more apparent than real.

The Type 2A strains detected in the Puerto Rican cohorts are all identical tothe Type 2A prototype sequence in the VP1 gene typing region, and the mini-mum-evolution phylogenetic analysis shows a close relationship of the twostrains analyzed to strains from Native Americans and a Japanese strain (Tokyo-1). There are several possible explanations for this high sequence similarity. First,there may have been relatively little time depth for these strains on the island. Thestrains may represent the Ostionoid population arriving from South America aftera.d. 600 rather than being derived from archaic island inhabitants arriving some-time after 4000 b.c. (Rouse 1948; Rouse 1993; Wilson 1999). Second, in this is-land settlement the founding population may have been small with significantfounder effects and other population bottlenecks. Third, unique mutations in theregulatory region may have affected virus strain survival. It is noteworthy that insix of eight of the Puerto Rican Type 2A regulatory regions that were sequenced,there was a deletion (nucleotides 53–62) similar to one seen in some East andCentral African strains (nucleotides 51–60) (Agostini et al. 1995; Chima et al.1998) and in Papua New Guinea (PNG-1A strains; nucleotides 52–61) (Ryschke-witsch et al. 2000). The fact that similar regulatory region deletions have arisen

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independently in different genotypes in different parts of the world suggests a se-lective advantage conferred within these populations. In Taino Indians the suc-cess of these Type 2A strains of JCV may have resulted in part from a selectiveadvantage due to the occurrence of this regulatory region mutation.

Maternally transmitted mtDNA and the paternally transmitted Y chromo-some are not reshuffled by recombination during cell meiosis, an important ad-vantage for their application in studies of human evolution and migration. DoesJCV enjoy the same advantage? Although there have been nearly 500 years of ge-netic admixture in New World Hispanic populations and thus direct competitionbetween JCV genotypes, presumably with some double infections, to date there isno evidence that strains of different genotypes have recombined in Puerto Rico.The two portions of the genome that were genotyped (VP1 gene and the regulato-ry region) are located on opposite sides of the genome and indicated consistentgenotype assignments. This is likely due to the inherent stability of the circularsupercoiled DNA genome of this virus. The exception that proves this rule is asingle isolate of JCV obtained in California, designated Type 5 (Jobes et al. 1998;Agostini et al. 1998d), which appears to represent a recombinant between theType 2B and Type 6 genotypes (Hatwell and Sharp 2000).

Our results are consistent with the trihybrid model for the peopling of Puer-to Rico and show that the major genotypes of JCV previously identified in Asia,Africa, and Europe can be identified in the present-day inhabitants of PuertoRico. The strains brought by the European settlers or the African slaves have beenmaintained largely unchanged for up to 500 years. The whole genomes of twoType 6 (African) strains were amplified and sequenced and shown to be mostclosely related to another Type 6 strain previously amplified from the brain of anAfrican American PML patient (Agostini et al. 1998c). The Type 3B strain wasvery closely related to a strain obtained from the urine of an African American inmainland USA (Agostini et al. 1997a). The Type 2A strains of the indigenouspeoples may have first entered the New World with Amerind ancestors6,000–12,000 years ago, but appear to have changed little in the intervening time.Importantly, all JCV strains obtained in Puerto Rico represent genotypes that hadpreviously been identified and described elsewhere. This suggests that the majorviral genotypes circulating in the native Caribbean population, in West and Cen-tral Africa, and in the Iberian peninsula at the time of Columbus, have been iden-tified.

In Puerto Rico the native Taino population, an Arawak tribe, were decimat-ed by warfare and disease, and had completely disappeared by the end of the 17thcentury, but the JCV gentoype (Type 2A) that their ancestors brought from Asiahas not only survived, but has actually thrived in their living descendants. Wesuggest that JCV genotyping can be a useful tool to reconstruct human populationhistory, even where the contribution of a founding population, such as the Taino,has long been obscured. The presence of particular virus strains in a populationmay emphasize a history lost or unappreciated. Conversely, those virus types thatare at a selective disadvantage may disappear over time when in direct competi-

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tion with more successful strains in the admixed population. As an example of thelatter, Type 6 strains were unexpectedly absent from an urban African Americancohort studied in Washington, D.C., although one was found in an African immi-grant living in the USA for 20 years (Chima et al. 2000).

JCV sometimes causes a fatal demyelinating disease, known as progressivemultifocal leukoencepalopathy (PML), in immunocompromised individuals. Pre-viously very rare, PML now develops in 4%–5% of AIDS patients in the USA.HIV-infected individuals who are also infected with JCV Type 2B appear to be athigher risk for PML (Agostini et al. 1998b). The low PML prevalence (0.8%) in alarge autopsy study of AIDS pathology in Brazil (Chimelli et al. 1992) is consis-tent with the general absence of Type 2B strains in Hispanic American popula-tions, and the predominance of Type 2A. It will be of interest to determinewhether the populations of other South and Central American or Caribbean coun-tries where Type 2A strains may predominate also have lower rates of PML inAIDS patients.

In conclusion, JCV genotypes from Asia, Africa, and Europe were all rep-resented in this Hispanic American cohort from Puerto Rico. However, viralstrain selection due to viral, genetic, and cultural factors appears to have led tounderrepresentation of the European and African components, while the NativeAmerican genetic element is overestimated. Thus, the proportions of Asian, Afri-can, and European genotypes of JCV do not reflect the quantitative genetic con-tributions of these population groups to the gene pool. It is possible, nevertheless,that in populations where these genetic contributions vary, as in the African pop-ulations of coastal Brazil (Krieger et al. 1965; Bortolini et al. 1999), the distribu-tion of JCV genotypes may vary accordingly.

Acknowledgments We thank Nyra Rodriguez for technical assistance. This work wassupported by the United States Public Health Service through grants G12RR/AI-03061and G12RR/AI03050 from the Research Centers in Minority Institutions (RCMI) Pro-gram, National Center for Research Resources, to the University of Hawaii at Manoa andthe Ponce School of Medicine, respectively. This study is a collaborative project of theRCMI Gateway Consortium.

Received 21 September 2000; revision received 7 December 2000.

Literature Cited

Agostini, H.T., G.R. Brubaker, J. Shao et al. 1995. BK virus and a new type of JC virus excreted byHIV-1 positive patients in rural Tanzania. Arch. Virol. 140:1919–1934.

Agostini, H.T., D.V. Jobes, S.C. Chima et al. 1999. Natural and pathogenic variation in the JC virusgenome. In Recent Res. Dev. Virol. Vol. 1, Part 3, S. G. Pandalai, ed. Trivandrum, India:Transworld Research Network, 683–701.

400 / fernandez cobo et al.

Agostini, H.T., C.F. Ryschkewitsch, G.R. Brubaker et al. 1997a. Five complete genomes of JC virusType 3 from Africans and African Americans. Arch. Virol. 142:637–655.

Agostini, H.T., C.F. Ryschkewitsch, E.J. Singer et al. 1998a. JC virus Type 1 has multiple subtypes:Three new complete genomes. J. Gen. Virol. 79:801–805.

Agostini, H.T., C.F. Ryschkewitsch, E.J. Singer et al. 1998b. JC Virus Type 2B is found more fre-quently in brain tissue of progressive mulitifocal leukoencephalopathy patients than in urinefrom controls. J. Hum. Virol. 1:200–206.

Agostini, H.T., C.F. Ryschkewitsch, and G.L. Stoner. 1996. Genotype profile of human polyomavirusJC excreted in urine of immunocompetent individuals. J. Clin. Microbiol. 34:159–164.

Agostini, H.T., C.F. Ryschkewitsch, and G.L. Stoner. 1998c. Complete genome of a JC virus genotypeType 6 from the brain of an African American with progressive multifocal leukoencephalopa-thy. J. Hum. Virol. 1:267–272.

Agostini, H.T., Y. Shishido-Hara, R.W. Baumhefner et al. 1998d. JC virus Type 2: Definition of sub-types based on DNA sequence analysis of ten complete genomes. J. Gen. Virol. 79:1143–1151.

Agostini, H.T. and G.L. Stoner. 1995. Amplification of the complete polyomavirus JC genome frombrain, cerebrospinal fluid and urine using pre-PCR restriction enzyme digestion. J. Neurovirol.1:316–320.

Agostini, H.T., R. Yanagihara, V. Davis et al. 1997b. Asian genotypes of JC virus in Native Americansand in a Pacific Island population: Markers of viral evolution and human migration. Proc.Natl. Acad. Sci. USA 94:14542–14546.

Alegria, R. 1974. Discovery, Conquest, and Colonization of Puerto Rico, 1493–1599. San Juan, Puer-to Rico: CEP.

Bertorelle, G., and L. Excoffier. 1998. Inferring admixture proportions from molecular data. Mol.Biol. Evol. 15:1298–1311.

Bofill-Mas, S., S. Pina, and R. Girones. 2000. Documenting the epidemiologic patterns of poly-omaviruses in human populations by studying their presence in urban sewage. Appl. Environ.Microbiol. 66:238–245.

Bortolini, M.C., W.A. Da Silva, D.C. De Guerra et al. 1999. African-derived South American popula-tions: A history of symmetrical and asymmetrical matings according to sex revealed by bi- anduni-parental genetic markers. Am. J. Hum. Biol. 11:551–563.

Chang, D.C., R.T. Tsai, M.L. Wang et al. 1996. Different genotypes of human polyomaviruses foundin patients with autoimmune diseases in Taiwan. J. Med. Virol. 48:204–209.

Chima, S.C., C F. Ryschkewitsch, K.J. Fan et al. 2000. Polyomavirus JC genotypes in an urban Unit-ed States population reflect the history of African origin and genetic admixture in modern Af-rican Americans. Hum. Biol. 72:837–850.

Chima, S.C., C.F. Ryschkewitsch, and G.L. Stoner. 1998. Molecular epidemiology of human poly-omavirus JC in the Biaka pygmies and Bantu of Central Africa. Mem. Inst. Oswaldo Cruz93:615–623.

Chimelli, L., S. Rosemberg, M.D. Hahn et al. 1992. Pathology of the central nervous system in pa-tients infected with the human immunodeficiency virus (HIV): A report of 252 autopsy casesfrom Brazil. Neuropathol. Appl. Neurobiol. 18:478–488.

Frisque, R.J., G.L. Bream, and M.T. Cannella. 1984. Human polyomavirus JC virus genome. J. Virol.51:458–469.

Guo, J., T. Kitamura, H. Ebihara et al. 1996. Geographical distribution of the human polyomavirus JCvirus type A and B and isolation of a new type from Ghana. J. Gen. Virol. 77:919–927.

Guo, J., C. Sugimoto, T. Kitamura et al. 1998. Four geographically distinct genotypes of JC virus areprevalent in China and Mongolia: Implications for the racial composition of modern China. J.Gen. Virol. 79:2499–2505.

Hanis, C. L., D. Hewettemmett, T. K. Bertin et al. 1991. Origins of United States Hispanics—Impli-cations for diabetes. Diabetes Care 14:618–627.

Hatwell, J.N., and P.M. Sharp. 2000. Evolution of human polyomavirus JC. J. Gen. Virol.81:1191–1200.

JC Virus in Puerto Rico / 401

Jimenez de Wagenheim, O. 1998. Puerto Rico: An Interpretive History from Pre-Columbian Times to1900. Princeton, NJ: Markus Wiener Publishers.

Jobes, D.V., S.C. Chima, C.F. Ryschkewitsch et al. 1998. Phylogenetic analysis of 22 completegenomes of the human polyomavirus JC virus. J. Gen. Virol. 79:2491–2498.

Jobes, D.V., J.S. Friedlaender, C.S. Mgone et al. 1999. A novel JC virus variant found in the Highlandsof Papua New Guinea has a 21-base pair deletion in the agnoprotein gene. J. Hum. Virol.2:350–358.

Kitamura, T., Y. Aso, N. Kuniyoshi et al. 1990. High incidence of urinary JC virus excretion in non-immunosuppressed older patients. J. Infect. Dis. 161:1128–1133.

Krieger, H., N.E. Morton, M.P. Mi et al. 1965. Racial admixture in north-eastern Brazil. Ann. Hum.Genet. (Lond) 29:113–125.

Lisker, R., R. Perezbriceno, J. Granados et al. 1986. Gene-frequencies and admixture estimates in aMexico City population. Am. J. Phys. Anthropol. 71:203–207.

Lisker, R., E. Ramirez, and V. Babinsky. 1996. Genetic structure of autochthonous populations ofMeso-America: Mexico. Hum. Biol. 68:395–404.

Long, J.C., R.C. Williams, J.E. Mcauley et al. 1991. Genetic variation in Arizona Mexican-Ameri-cans—Estimation and interpretation of admixture proportions. Am. J. Phys. Anthropol.84:141–157.

Merriwether, D.A., S. Huston, S. Iyengar et al. 1997. Mitochondrial versus nuclear admixture esti-mates demonstrate a past history of directional mating. Am. J. Phys. Anthropol. 102:153–159.

Morales Carrion, A. 1983. Puerto Rico: A Political and Cultural History. New York, NY: W.W.Norton.

Morner, M. 1967. Race Mixture in the History of Latin America. Boston, MA: Little, Brown. Ou, W.-C., R.-T. Tsai, M. Wang et al. 1997. Genomic cloning and sequence analysis of Taiwan-3 hu-

man polyomavirus JC virus. J. Formos. Med. Assoc. 96:511–516.Padgett, B.L., D.L. Walker, G.M. ZuRhein et al. 1971. Cultivation of papova-like virus from human

brain with progressive multifocal leucoencephalopathy. Lancet 1:1257–1260.Rouse, I. 1948. The Arawak. In Handbook of South American Indians. Vol. 4, The Circum-Caribbean

Tribes, J.H. Steward, ed. Washington, D.C.: Smithsonian Institution, Bureau of American Eth-nology, 507–546.

Rouse, I. 1993. The Tainos: Rise & Decline of the People Who Greeted Columbus. New Haven, CT:Yale University Press.

Rout, L.B. 1976. The African Experience in Spanish America: 1502 to the Present Days. Cambridge,England: Cambridge University Press.

Ryschkewitsch, C.F., J.S. Friedlaender, C.S. Mgone et al. 2000. Human polyomavirus JC variants inPapua New Guinea and Guam reflect ancient population settlement and viral evolution. Mi-crob. Infect. 2:1–10.

Sans, M. 2000. Admixture studies in Latin America: From the 20th to the 21st century. Hum. Biol.72:155–177.

Santos, S.E.B., F.M. Salzano, M. Helena et al. 1983. Mobility, genetic markers, susceptibility tomalaria and race mixture in Manaus, Brazil. J. Hum. Evol. 12:373–381.

Shah, K.V., R.W. Daniel, H.D. Strickler et al. 1997. Investigation of human urine for genomic se-quences of the primate polyomaviruses simian virus 40, BK virus, and JC virus. J. Infect. Dis.176:1618–1621.

Shriver, D.M. 1997. Ethnic-affiliation estimation by use of population-specific DNA markers. Am. J.Hum. Genet. 60:957–964.

Stoner, G.L., H.T. Agostini, C.F. Ryschkewitsch et al. 1998. JC virus excreted by multiple sclerosispatients and paired controls from Hungary. Mulitple Sclerosis 4:45–48.

Sugimoto, C., T. Kitamura, J. Guo et al. 1997. Typing of urinary JC virus DNA offers a novel meansof tracing human migrations. Proc. Natl. Acad. Sci. USA 94:9191–9196.

Swofford, D.L. 2001. Paup 4.0 User’s Manual: Phylogenetic Analysis Using Parsimony. Sunderland,MA: Sinauer Associates.

402 / fernandez cobo et al.

Wilson, S.M. 1999. The Indigenous People of the Caribbean. Gainesville, FL: University Press ofFlorida.

Yogo, Y., T. Iida, F. Taguchi et al. 1991. Typing of human polyomavirus JC virus on the basis of re-striction fragment length polymorphisms. J. Clin. Microbiol. 29:2130–2138.

Yogo, Y., T. Kitamura, C. Sugimoto et al. 1990. Isolation of a possible archetypal JC virus DNA se-quence from nonimmunocompromised individuals. J. Virol. 64:3139–3143.