EMERGING INFECTIOUS DISEASES, Vol. 1, No. 3, July-September, 1995


DISPATCHES

HIV-1 Patients May Harbor Viruses of Different Phylogenetic
Subtypes: Implications for the Evolution of the HIV/AIDS Pandemic

The virus variants isolated from HIV-infected persons worldwide
share remarkable diversity, especially in the envelope
glycoprotein, gp120. Phylogenetic studies have clustered HIV-1
isolates into eight subtypes (A-H). Nevertheless, even within a
single infected person, HIV is present as a "quasi-species," or a
swarm of closely related variants. This genetic diversity, which
in the case of HIV-1 accumulates at a rate of approximately one
nucleotide substitution per genome per replication cycle, gives
the virus an enormous flexibility to respond to a wide array of
in vivo selection pressures. As a consequence, drug-resistant and
immunologic escape mutants are rapidly generated in infected
persons through all stages of infection. On a global scale, the
HIV pandemic is recognized as consisting of many separate
epidemics, each with characteristic geography, affected
populations, and predominant viral strain type. With an estimated
15 million infected persons, the geographic distribution of viral
subtypes is becoming more dispersed, and these demarcations are
further confounded by growing evidence of mixed infections.

The epidemic emergence of mixed heterotypic infections with HIV-1
and HIV-2 variants has been recognized for some time in the
geographic areas where both types of viruses are present. We
reported these infections in C“te d'Ivoire and Brazil (1,2);
they have also been reported from India (3). In contrast,
homotypic mixed infections of distinct HIV-1 variants have only
recently been suggested by the presence of broadly reactive sera
and evidence of HIV recombinants from geographic regions in which
multiple HIV-1 subtypes are circulating. Dual HIV-1 infection in
two patients from Thailand has been demonstrated by viral DNA
sequence analysis (4).

As the HIV-1 pandemic has grown, the simultaneous presence of
multiple subtypes in a region has become common. As a
consequence, an increased frequency of HIV-1 mixed infections
could be expected. Thus, there is a need to estimate the
prevalence and geographic distribution of this type of infection.
Sequence analysis of HIV proviral DNA has been the method of
choice to characterize HIV genetic diversity. However, because
even relatively limited sequence determinations of small
polymerase chain reaction (PCR)fragments are time-consuming and
very labor-intensive, this method is not particularly practical
for large-scale molecular epidemiologic studies. To address this
problem, we have developed a genetic method based on restriction
site polymorphism to screen for homotypic HIV-1 mixed infections
within infected populations. The concept of this assay is based
on the observed correlation between the restriction maps of HIV-1
isolates with their phylogenetic classification, which is based
on the sequence data. Thus, certain restriction enzymes may be
used to predict the phylogroup of HIV-1 infected samples. The
differences in electrophoretic mobility of endonuclease
digestion products result from restriction site polymorphisms in
the selected region of the HIV-1 genome and allow for quick
recognition of the distinct phylogenetic subtypes. A 297 bp pol
fragment spanning the entire viral protease gene is used for our
analysis. The viral gene is amplified by nested PCR using DNA
templates from uncultured peripheral blood mononuclear cells
(PBMC) or virus culture. Preliminary classification of HIV-1
strains to well defined subtypes A, B, C, D, and F is done by
sequential endonuclease restriction analysis. AluI restriction
polymorphism in a PCR-amplified protease gene segregates viral
strains into two groups: subtypes B and D belong to one group,
and subtypes A, C, and F to another (Figure 1A cannot be viewed 
in ASCII). Further differentiation of HIV-1 subtypes within those 
two groups is accomplished by analysis of HinfI, BclI, MaeI, 
SpeI, an ScaI restriction enzyme digestion patterns of the 
protease gene (Pieniazek et al., manuscript in preparation). 
The electrophoretic migration patterns visualized by ethidium 
bromide staining or by radiolabeled probes are then determined 
on a 10% acrylamide gel. In single infections, a single 
restriction pattern is detected, whereas in multiple infections 
involving HIV-1 strains of distinct subtypes, complex digestion 
patterns are observed in infected persons. As an example, in 
Figure 1A, we present three distinct AluI restriction patterns 
of the protease gene that are characteristic for single 
infections by viruses of subtypes A, C, and F (pattern #1) 
and by subtypes B and D (patterns #2 and #3). In Figure 1B, 
we show a typical combination of two distinct AluI restriction 
patterns (#1 and #2) found in a patient infected with two 
viral strains of subtypes F and B. Basing our analysis on 
the conserved protease gene region, we should detect most 
HIV-1 strains; however, some highly divergent isolates could 
escape PCR amplification as a result of primer mismatches. 
Moreover, since a single nucleotide substitution could either 
generate or destroy a restriction site, sequence analysis 
remains the ultimate tool in identifying variants of multiple 
infections. Nevertheless, this assay can be conveniently
applied to screen a large number of samples.

By using this method, we have screened HIV-1 proviral DNA from
208 specimens collected from countries in South America, Africa,
and Asia where HIV-1 strains of distinct subtypes are found. We
observed the simultaneous presence of two distinct digestion
patterns in PCR amplified protease gene (Figure 1B cannot be viewed
in ASCII) in 31 samples; our observation suggests 
superinfection with HIV-1 strains of distinct origin. 
To eliminate the potential for laboratory cross-contamination, 
we analyzed the restriction patterns of the protease gene 
from multiple aliquots of the patient's PBMC. 
In addition, the analysis was repeated on DNA
from a second collected blood sample from each of the patients.
The analyses for the first five of 31 patients were completed,
and data are summarized here (details are in Janini et al.,
manuscript in preparation). Sequence and phylogenetic analysis of
the viral protease gene (Figure 2 cannot be viewed in ASCII.) in PBMC from those five
patients confirmed dual infections caused by HIV-1 strains of
subtypes B and F in one person (Br5), subtypes F and D in another
patient (Br22), and subtypes C and D in a married couple (Br19
and 20). Moreover, in the child (Br30) of this couple, two
distinct AluI digestion patterns were also found; the major
HIV-1 strain clustered among subtype C viruses of the parents.
The minor strain of this child is likely to represent subtype D,
but there was not sufficient material for cloning and further
sequencing of this strain.  

Detection of naturally occurring heterotypic and homotypic
multiple infections may have important implications for
immunotherapies because infection with one HIV subtype may not
fully protect against subsequent superinfections with distinct
HIV strains. However, we do not know if the acquisition of
viruses in the dually infected adult patients was sequential or
simultaneous. Nevertheless, the consequences of mixed infections
may profoundly affect the ability of the virus to change and may
modify the direction of the pandemic through altered patterns of
viral pathogenesis, increased genetic variation through
recombination, and the generation of pseudotype virions,
including phenotypically mixed virus particles. It is to be
anticipated that such events would ultimately broaden the
cellular tropism for HIV and mandate the designed polyvalent
immunotherapies. Finally, our data together with recently
published genetic analysis for HIV-1 and HIV-2 (5) suggest that
multiple homotypic infections with divergent HIV strains may be
more common than previously thought. The screening assay
described here will be useful in estimating incidences of
such HIV-1 infections. We believe that this information is
crucial for both evaluating the pandemic and developing
intervention strategies.

Danuta Pieniazek,* Luiz M. Janini,*#  Artur Ramos,*#  Amilcar
Tanuri,# Mauro Schechter,& Jose M. Peralta,# Anna C.P. Vicente,
Norman J. Pieniazek,* Gerald Schochetman,* and Mark A. Rayfield*
*National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia, USA; #Universidade
Federal do Rio de Janeiro, and &Hospital Clementino Fraga Filho,
and Instituto Oswaldo Cruz, Rio de Janeiro, Brazil

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