DEMANDA DE LOS CONSUMIDORES RESIDENCIALES

Serological studies already conducted of children and family groups in several

parts of Africa (Mayama et al, 1998; Gessain et al, 1999; Plancoulaine et al,

2000) point to significant intra-familial transmission between mothers and their

children and between siblings. To date, no studies have been done using

molecular techniques to confirm whether such transmission routes exist. In the present study, Malawian family groups in which one member had KS were selected to investigate modes of familial KSHV transmission.

To effect the study, two regions of the hypervariable ORF K l, designated

K l/V l and K1/V2, were chosen for amplification and sequencing. These

regions were selected as their variable nature facilitates the best possible

comparison between sequences. From a total of 22 families, 2 or more

sequences could be compared in 8 families. In 4 families, identical K l/V l sequences were recovered, and in 5 some diversity was present. Both similar and divergent sequences could be recovered from one family, E. These data suggest that in families B, G, K and Z, intra-familial transmission events have

taken place. In families W, X, T, E and B non-identical sequences were

revealed between family members suggesting extra-familial transmission events occurred. However, not all of the family members were infected with KSHV

and their genomes were not available for sequence comparison. In some

instances, the mother, who may be considered the most likely source of infection, was not available for study. Within the two predominant subtypes, A5 and B, sequences between family groups were very similar. K l subtypes appear to be highly conserved within certain ethnic populations and across geographical

regions (Zong et al, 2002), so while family groups may not live in the same locality, they may have carried closely related viral variants.

The degree of nucleotide variation in many families was less than 2%, although the same sequence for each sample was consistently detected in multiple clones. Nevertheless, a proofreading polymerase was not used to amplify ORF K l/V l and K1/V2 from the samples. Multiple clones were taken and analysed from a single PCR reaction and if a polymerase induced error occurred early in PCR cycling, it would represent a significant percentage of the final pool of

amplicons. Sequencing only three to five of these amplicons would not

necessarily ensure that the true consensus sequence was obtained.

KSHV DNA could not be amplified from the blood of any KSHV antibody positive family member, but was amplified in a high percentage of their mouth rinse samples. As this study and others have shown, children are frequently

infected with KSHV in endemic regions like Malawi (Angeloni et a l, 1998;

Mayama et a l, 1998; Olsen et al, 1998; Andreoni et al, 1999; Gessain et al,

1999; Plancoulaine et al, 2000; Davidovici et al, 2001). A nonsexual route of

transmission, facilitated by close contact with the saliva of an infected

individual, may explain why KSHV is acquired during childhood. KSHV

transmission may thus be similar to EBV transmission in underdeveloped regions: EBV is principally acquired before 3 yrs of age through close contact

with infected saliva (Yao et al, 1985).

A previously described PCR RFLP method (Zhang et al, 2000) was adapted to

make further comparisons of KSHV genome relatedness between family members. For this, another region of the KSHV genome, the IRD of ORF 73 was examined. Using the PCR RFLP technique, 8 families, 6 of which had been analysed by ORF K l sequencing, and 5 individual samples were studied. The RFLP method confirmed the data obtained by K l/V l sequencing in 5 of these 6

families. Study individuals in family G revealed dissimilar RFLP patterns

however the father G2 and son G l carried identical ORF K l/V l sequences. Samples from study subject G (mother of G l and wife of G2) could not be

amplified for ORF K l/V l but yielded a different RFLP pattern from both her son and husband. Previous studies suggest that RFLP patterns are unique to an

individual and are invariant in cell lines passaged over time (Zhang et a l, 2000).

Therefore, a comparison between two individual samples may be considered accurate and specific and members of family G can be said to carry different KSHV genomes based on the RFLP results. The polymorphic nature of the IRD

region (Gao et a l, 1999) allowed sequence variation between samples to be

readily identified, however it did not provide the specific sequence information gathered from PCR sequencing. A proofreading polymerase was not used and errors generated due to the high G+C content of the IRD region and areas of multiple repeated sequence may have created the apparent differences in RFLP pattern.

Further work: KSHV transmission is clearly very complex in Malawi,

involving both familial and community wide routes of infection. In this study, 8 families could be studied by direct sequence comparison of KSHV genomic regions. It is apparent that routes of transmission other than from mother to child are involved. Further clarification of KSHV transmission routes may be achieved by studying a larger sample size than that described here. In addition, significant data have been gathered about chimeric genomes and K1 subtype

linkage across the genome (Zong et al, 2002). Gathering such information

could allow more specific comparisons to be made between viral sequences recovered from various family members.