Non-clade B virus: epidemiology, testing strategies and resistance
Yasmin Halima, European AIDS Treatment Group
Given the plethora of information shared at meetings such as these, it is easy sometimes to forget that the fervent presentation of scientific expertise, treatment advances and diagnostic developments serve to further elucidate our understanding of the experiences of people infected by the HIV clade-B virus. Non-B subtypes have been much less characterised, including the widespread prevalence of A, C, D and E clades in developing nations.
The heterogeneity of the HIV virus, and the fact that most of the infected populations live outside of the western world, presents its own epidemiological, human and political challenges. It is inspiring therefore to note a more robust body of evidence emerging, largely in response to the evolving diversity of population groups within the western world, reflective of our patterns of migration, commerce and travel.
The changing viral landscape
Studies to map population genetic characteristics of non-clade B strains were discussed in a number of prospective and retrospective analysis of testing cohorts. Most notable of these were two studies from the New York City Department of Health (NYCDOH). The first study collected 230 samples between 1994 and 1999, 196 of which were from persons born in Asia, Africa and South America.  Phylogenetic analysis revealed that of these, 55% had viral variants similar to the typical Northern American subtype B viruses. However, nearly 30% were found to be infected with subtype A, 6% with subtype G and nearly 4% were classified as subtype C.
Other variants less frequently present included subtypes E, F, D and H virus. A second retrospective study from a similar immigrant population base, found that from 992 positive specimens screened, 600 were reactive to non-B peptides.  Of these, 237 were subtype C reactive, 166 were subtype A, 29 were subtype D, 11 were subtype E and 7 were subtype F. 120 samples were found to present mixed subtypes of which 30 could not be typed. The researchers also found 56 samples that were reactive for HIV-2. The authors conclude from both studies that ‘public health officials and practising physicians should be aware of the growing genetic diversity of HIV-1 in this country, particularly in areas with a sizeable immigrant population.’
Crossing the boundaries
Further confirmation that behaviour particularly amongst high-risk groups, influences the prevalence of genetic variations even across geographical borders was presented in some interesting epidemiological studies from the Indo-Chinese border. From genetic subtyping, the first of these studies revealed that subtype A/E recombinant virus predominates in all at-risk populations throughout Vietnam.  Interestingly, the subtype E identified between two provinces in northern Vietnam were found to be closely related phylogenetically, to those found in the neighbouring Guangxi province in China.
They shared the unique valine substitution at position 12 on the V3 loop. This particular variant, uniquely found among IDUs on the China-Vietnam border has now been classified as subtype ‘Ev’. A chronological assessment further ‘indicated that the HIV outbreak among IDUs in northern Vietnam was caused by a recent introduction of a highly homogenous subtype E variant (Ev) of similar origin to the virus prevailing in nearby province of southern China’.The second study between the Guangxi Health and Anti-Epidemic Center, China, and John Hopkins in Baltimore, corroborate these findings with their own epidemiological and virological analysis of IDUs in this region. 
They link the sharing of injecting equipment and unprotected sex amongst this group as significantly associated with transmission of infection. They conclude therefore, that ‘designing and implementing effective intervention strategies targeted towards both injection drug users and high risk sexual behaviour are urgently needed to further reduce HIV-1 spread in China’.
Beyond the prevention truism, although we have known of the link between high-risk behaviour and transmission, this study provides important confirmation of how viral spread is influenced by an evolving demography and group behaviour. Of greater concern is that as we see increasing evidence of global mobility, this will undoubtedly be reflected in the expansion of viral diversity.
The challenge within HIV is not only to strengthen scientific development in the mapping of this evolution, but also to sustain an aggressive awareness on the need for prevention amongst individuals and communities most affected.
The A, B, C of resistance
Limitations to our knowledge of non-clade B extends to the understanding of resistance patterns and their treatment impact on people infected with non-clade B virus. ‘Resistance mutations until now characterised were found in B-subtype virus of developed countries’, as Virco reminded us at the meeting. This assertion was supported by an impressive study based on data from their equally impressive database, yielding an interesting presentation on non-clade B resistance.  In partnership with a number of hospitals in Brazil, Virco analysed resistance mutation profiles from non-B and more divergent B-subtype viruses through ex vivo phenotyping, ‘in order to establish an exact correlation between the genotyping data and the clinical management counselling for those exotic [sic] virus subtypes’.
Two groups were stratified into virologic responders and non-responders and proviral and plasma viral genomes were sequenced and subtyped. Individuals from the non-B category were strongly associated with non-responder group and were more likely to progress rapidly to resistance after HAART. They also found in this group, a positive correlation between time under HAART and lack of response to ART. Interestingly, non-B subtypes did not present the L90M and I84V mutations and instead, used mainly G48V and V82A/F to achieve drug resistance; this combination of resistance mutations was associated with strong cross-resistance to all four PIs in those with the non-B virus. They conclude therefore that, ‘these findings suggest that non-B subtype HIV-1 strains are less susceptible to HAART, and use a different mutation pattern when compared to B isolates. This fact will impact on the interpretation of the HIV genotypic tests when non-B isolates are analysed and pose a major concern over the efficacy and lasting of combined anti-HIV therapy in countries which epidemic is driven by non-B subtype HIV-1 strains.’
In the unfolding scenario of population and viral heterogeneity, the issue of screening and testing strategies become paramount. A collaborative venture between the CDC, Atlanta and Thai health authorities (Parekh, B et al) explored a ‘testing algorithm to detect recent infections among persons infected with HIV-1 subtype B or E in Thailand’. 
From longitudinal specimens collected from 90 incident infections among IDUs in Bangkok, they calculated the ‘window period’ (time between incident of infection and seroconversion) on the sensitive and less-sensitive tests (3A11-LS assay). Another 114 specimens were collected from patients with AIDS having less then 200 CD4 cells who were also infected with subtype E. They found that recent infections within the subtype B group could be identified using this strategy, with a window period of 155 days. However, this window period within the subtype E individuals was extended to around 270 days. Worryingly, 8% from the subtype E group with AIDS-defining illnesses and around 4% of from the same E group who had been long-term infected were misclassified as recent infections.
The conclusion drawn is that the current 3A11-LS assay has a different window period for detecting recent infections among people in Thailand infected with either subtype B or E. Estimation of HIV-1 incidence using this testing strategy in populations with non-B subtype prevalence may require modification of the window period and/or cut-off value’. This means therefore that the ‘development of an assay with similar performance characteristics among different subtypes for detecting recent HIV-infection would be highly desired’.
Sero-subtyping at the Royal Free Hospital in London suggests a prevalence of 15-20% non-clade B HIV-infections in their clinic population. Aside from epidemiological concerns there are implications for treatment. Diminished sensitivity to PIs has been observed (in-vitro) for some non-clade B HIV-1 (Descamps D et al. AIDS. 1998 12(9):1109-11). Resistance testing (both geno and phenotyping) may also be difficult as fragment amplification has less success with non-clade B samples.
- Lin HH et al. Genetic characterisation of HIV-1 strains in an immigrant population living in New York City. 7th CROI; January 30-February 2, 2000; San Francisco, California. [Abstract 170].
- Beatrice ST et al. Detection of large numbers of HIV-1 group M non-B subtypes in New York City. 7th CROI; January 30-February 2, 2000; San Francisco, CA. [Abstract 171].
- Takebe Y et al. Cross-border spread of closely related HIV-1 subtype E variant among injecting drug users (IDUs) in Chino-Vietnam boundary. 7th CROI; January 30-February 2, 2000; San Francisco, CA. [Abstract 179].
- Beyrer C et al. An emerging HIV epidemic among injecting drug users in Guangxi province, Southern China. 7th CROI; January 30-February 2, 2000; San Francisco, CA. [Abstract 180]
- Caride E et al. Genotyping and phenotyping analysis of B and non-B HIV-1 subtypes from patients under HAART. 7th CROI; January 30-February 2, 2000; San Francisco, CA. [Abstract 745].
- Parekh B et al. Evaluation of a sensitive/less-sensitive testing algorithm to detect recent infection among persons infected with HIV-1 subtype B or E in Thailand. 7th CROI; January 30-February 2, 2000; San Francisco, CA. [Abstract 767].