Use of molecular epidemiology for investigation of transmission cases is often complicated by the fact that the determination always is very difficult, since the molecular analysis is performed on the HIV-1 virus in samples obtained with different time spans from the estimated time of transmission that vary from case to case. The HIV-1 variation also evolves individually depending on the individual patient and the course of infection.
The fact that the estimated time of transmission of HIV-1 in this study was more than 4 years prior to the discovery of the HIV-1 infection of the child makes the investigation of the molecular epidemiology more difficult and the interpretation of the phylogenetic analyses harder. Because of these difficulties, we selected three different genes for our analysis (gag, pol, and env). By doing so we tried to bypass any randomly phylogenetic relationship in a certain gene that might have disturbed the investigation. In choosing this approach, establishing a genetic relation was further challenged and demanded a true genetic relationship between the man's virus and the child's virus, which would be elucidated by the phylogenetic analyses. In addition, three different phylogenetic tools were used to analyze the genetic relatedness of HIV-1 from the two subjects: NJ, ML, and MP. However, it is important to note that determination of the transmission direction between the child and the man could not be resolved by these molecular investigations due to the fact that either of these individuals could have been infected by a third party. This molecular investigation should only be used in conjunction with additional evidence.
The data on sequence divergence from the described genes (gag, pol
, and env
) showed that the divergence differed depending on the gene in question. Other investigations on related transmission cases have shown that the divergence differs from case to case (4
). We have previously shown that the DNA sequence of the V3-loop was conserved during the first 24 weeks of infection in a donor-recipient pair (18
). In a study by Bobkov et al. it was found that the interpatient variation ranged from 5.9 to 6.6% (4
). Belec et al. showed in an investigation of intrafamilial transmission that the divergence in the C2-V3 region was between 1.2 and 5.0% (2
Goujon et al. investigated a transmission case analyzing the pol
). They found a very high degree of divergence depending on the sequences in question. They further showed that the divergence ranged from 11.33% within group M to 26.37% when group O was included, which shows that reference sequences and control sequences should be selected carefully. Our results for the pol
gene show that related sequences can differ substantially.
A previous investigation (14
) concerning a nosocomial HIV transmission shows, in comparison with the present study, that the p17gag
genes can differ substantially and still be phylogenetic related. Katzenstein et al. found that the p17gag
genes of two individuals differed by 0.9% (14
), while the man and the child differed by 2.66 to 2.95%. Of note is that the two transmission cases have different time spans from infection to discovery (i.e., the point at which patient samples were obtained). This can explain the difference in divergence between the transmission cases investigated, together with the fact that virus infection evolves in a fashion specific to each infected person, depending on the immunological status of that person and the nature of the virus.
The results from the alignment of the C2-V3-C3 region showed that the divergence was higher in this gene compared to p17gag
. Blanchard et al. found in their investigation of a nosocomial transmission from a surgeon to a patient that the gag
sequence divergence was higher compared to the env
). This was, however, caused by the fact that the individuals were infected by a recombinant HIV-1 A/F subtype.
Because the V3 region has been described as the principal neutralizing epitope, many studies have investigated the variation within the envelope C2-V3-C3 region (7
). This has led to speculation on how suitable this region is in resolving transmission cases (11
). Leitner et al. investigated which regions were most accurate in the reconstruction of a true transmission case by using phylogenetic methods on sequences derived from the p17gag
region or the C2-V3-C3 region and sequences from the two regions in combination (15
). These authors found that the most accurate phylogenetic tree compared to the true tree was constructed by using sequences from p17gag
and C2-V3-C3 in combination, and they also found that analysis of the C2-V3-C3 region was more accurate than analysis of the p17gag
When the time spans from the estimated time of infection to the collection of blood samples in this study (21 months and 3 years respectively) were taken into account, the phylogenetic analysis using either p17gag or p17gag plus C2-V3-C3 showed more accurately the time-dependent relationship between the sequences compared to the analysis using the C2-V3-C3 region alone.
The phylogenetic analysis of the pol gene confirmed the relatedness of the two individuals viruses.
The one-amino-acid deletions in the C2-V3-C3 region provided a very strong epidemiological link between the two patients, and we were not able to detect this deletion in any of the sequences from the Los Alamos database that we included in this study.
In conclusion, the results from the phylogenetic analyses, the sequence distances between the virus from the man and the virus from the child, and the identification of the unique molecular fingerprint in the env gene together indicate that the virus from the man and that from the child were epidemiologically linked.