Human Herpesvirus 8 Genotype E in Patients with Kaposi Sarcoma, Peru

To determine human herpesvirus 8 (HHV-8) K1 genotypes in patients with Kaposi sarcoma (KS) from Peru, we characterized HHV-8 in 25 KS biopsy samples. Our findings of 8 A, 1 B, 14 C, and 2 E subtypes showed high HHV-8 diversity in these patients and association between E genotype and KS development.

Recent studies demonstrated that classic KS is common in Peru and that AIDS-KS incidence is increasing because of the spread of HIV infection (10,11). Classic and epidemic KS occurred in patients of Amerindian origin (Quechuas) and in mestizos, refl ecting the multiethnic origin of the Peruvian population.
A goal of our study was to determine the HHV-8 genotypes for a series of classic KS or AIDS-KS cases in Peru. We also aimed to report KS in patients infected by an E subtype.

The Study
We studied a series of 36 KS tumors diagnosed during 1989-2002 at the Hospital Nacional Cayetano Heredia in Lima. All these formalin-fi xed, paraffi n-embedded biopsy samples were stained by using hematoxylin-eosin stain and Perl methods. Immunohistochemistry was performed on deparaffi nized sections by using monoclonal antibodies directed against CD34 and latent nuclear antigen (LANA-1) (12).
Histopathologic analysis was originally conducted on 36 biopsy samples, mostly from skin, diagnosed as KS. Three patterns were observed by hematoxylin-eosin stained specimens. The fi rst was characterized by dilated, irregular, and angulated blood vessels in the dermis, associated with a variable number of lymphocytes. In the second pattern, dermal vascular channels lined by plump spindle cells were seen; some of these spindle cells coalesced to form aggregates, which were poorly delineated and often located around blood vessels. The third pattern (nearly half of all biopsy samples) was characterized by well-delineated sheets and bundles of spindle cells, which coalesced to form nodules. The proportion of spindle cells labeled with the monoclonal antibody LANA-1 varied according to the histopathologic pattern; the bundles and sheets of spindle cells in the third pattern displayed the strongest signal ( Figure 1, panel A).
DNA was extracted from the 36 formalin-fi xed, paraffi n-embedded biopsy samples; DNA quantity and quality were appropriate in 30 biopsy samples. A faint PCR signal of the expected size was seen after single PCR in 12 samples for VR1 (VR1-outer) and in 4 cases for VR2 (VR2-outer) amplifi cation. After nested PCR, a signal was obtained in 25 cases for VR1-inner and in 17 cases for VR2-inner (online Appendix Table, www.cdc.gov/eid/ content/16/9/1459-TechApp.htm). After cloning and sequencing procedures, the HHV-8 genotype was obtained for 25 different KS cases, of which 8 genotypes belonged to the A subtype, including an A5, and 14 belonged to the C subtype. An E subtype was identifi ed for 2 patients. In 1 case, a B subtype was determined. Among these 25 sequences, 16 were unique and 9 formed 3 groups of identical sequences. The 25 VR1-inner sequences exhibited 0%-24.4% nt divergence and 0%-43.1% aa acid divergence among pairwise comparisons. When the VR1-outer and VR2-inner sequences (536 bp) of the 2 E subtype strains in Peru were combined, the nucleotide divergence was ≈7% and reached 10% at the amino acid level. The 04480 and 06772 E subtype strains were closer to the Brazilian Amerindian strains (Kat, Sio, Wai, Tir, Tupi) than to the Ecuadorian (Hua1, Hua2, Hua3) or French Guianan Amerindian (Wagu) strains.
Phylogenetic analyses were performed by using 2 sets of sequences; 25 VR1-inner ( Figure 2) and 11 VR1outer sequences (data not shown). Forty-seven prototype strain sequences were added. The main molecular HHV-8 subtypes, A-E, were identifi ed on the basis of consistent topology and bootstrap values obtained (Figure 2; data are not shown for other phylogenetic analyses performed, for example with the 11 VR1-outer sequences obtained after the fi rst round of PCR). Among the 25 VR1-inner sequences, 22/25 were located in the large A/C subtype, and 8 strains belong to the A subtype, with strains scattered among 3 different subgroups (Figure 2). The 06758 strain belongs to the typical sub-Saharan A5 group. Fourteen strains are distributed among different groups in the C subtype, and the remaining sequences clustered in the sub-Saharan African B (04489) and Amerindian E (06772 and 04480) subtypes.
Two AIDS-KS mestizo patients ( Figure 1) were thus found to be infected by typical E subtype HHV-8 strains: a 51-year-old man with a tumor on his neck and a 24-yearold man with multiple tumors on the upper limbs. These tumors were mostly macroscopic nodules but displayed major histologic differences (Figure 1). CD34 (data not shown) and LANA-1-positive cells (Figure 1) were noted in both KS biopsy samples.

Conclusions
HHV-8 K1 gene characterization of KS tumor biopsy samples demonstrated high molecular genotype diversity, including 4 of the 5 main known molecular subtypes. The most frequent were the A and C subtypes, typically of European origin. Other patients were infected by a sub-Saharan African HHV-8 genotype (1 A5 and 1 B) or by HHV-8 genotype E strains of Amerindian origin.
Such fi ndings were not unexpected because persons in Peru are of many ethnicities. Indeed, the genetic background of the Peruvian population is diverse, refl ecting the multiple waves or populations that colonized the country during the last millennium. Schematically, this began with different Amerindians groups in the Lithic period (infected possibly with E subtype), followed by the Spanish colonization of the Americas mainly from southern Europe (infected possibly by A and C subtypes) and later slave trade from Africa (infected by A5 or B subtypes). Such HHV-8 strain diversity has been previously observed in French Guiana (6,14).
HHV-8 genotype E previously has been found exclusively in Amerindians from Central and South America, including Brazil (5,9), Ecuador (7), and French Guiana (6). In each instance, HHV-8 was detected in blood samples, and there was some debate about the presence/development of KS in such highly infected populations (15).
We demonstrated that KS can occur in HHV-8 subtype E-infected persons. Indeed, in 2 AIDS-KS patients, an E genotype was characterized in the tumor lesions. Further studies are ongoing to provide new insights into the distribution and genetic epidemiology of such HHV-8 infection in Amerindian populations.  Figure 2. Unrooted phylogenetic tree generated with the neighborjoining method (PAUP* version 4.0b10; http://paup.csit.fsu. edu) on the best 165-bp alignment of the variable region [VR] 1 comprising 79 human herpesvirus 8 nt sequences, including 25 novel sequences generated (GenBank accession nos. GU827339-GU827363). The strains were aligned with Data Analysis in Molecular Biology software (http://dambe.bio.uottawa.ca/software. asp), and the fi nal alignment was submitted to the ModelTest software version 3.6 (http://darwin.uvigo.es/software/modeltest. html) to select, according to the Akaike Information Criterion, the best model to apply to phylogenetic analyses. The selected model was the general time reversible model. The reliability of the inferred tree was evaluated by bootstrap analysis on 1,000 replicates. Bootstrap support is noted on the branches of the tree.