| |
HIV Existed 100 Years Ago Study Reports, Urbanization of Africa Spread HIV
(3 Nature articles below)
|
| |
| |
The urbanization of Africa in the early part of the 20th century may be partly responsible for the spread of HIV. The suggestion was derived from an analysis of a tissue sample taken in 1960 in what was then Leopoldville, the colonial capital of the Belgian Congo, according to Michael Worobey, D. Phil., of the University of Arizona, and colleagues.
HIV RNA taken from the sample, which had been fixed in paraffin, provided the second oldest viral sequence, next to one from a blood sample -- also from Leopoldville -- taken in 1959.
The key finding is that the two samples -- both of HIV-1 Group M -- are sufficiently different that their earliest common ancestor must have existed about 50 years previously.
At that time, Leopoldville -- and most population centers in central Africa -- was small, providing only a small pool in which HIV could spread. But as the cities grew, HIV diversified, the researchers suggested. medpagetoday.com
"The interpretation that HIV-1 was spreading among humans for 60-80 years before AIDS was first recognized should not be surprising. If the epidemic grew roughly exponentially from only one or a few infected individuals around 1910 to the more than 55 million estimated to have been infected by 2007, there were probably only a few thousand HIV-infected individuals by 1960, all in central Africa. Given the diverse array of symptoms characteristic of AIDS, and the often-long asymptomatic period following infection, it is easy to imagine how the nascent epidemic went unrecognized. Conversely, such a low prevalence at that time implies that the Congolese co-authors of the paper2 were very lucky to come across this infected sample, even if most infections were concentrated in the area of Leopoldville. But can we trust these sequences?"......it seems likely that all of the early diversification of HIV-1 group M viruses occurred in the Leopoldville area......Leopoldville was not only the largest of these cities, but also a likely destination for a virus escaping from southeast Cameroon. In the early 1900s, the main routes of transportation out of that remote forest region were rivers; those surrounding this area flow south, ultimately draining into the Congo River, and leading to Leopoldville.....The date estimates of Worobey et al. are for an ancestral virus, present in the first individual to give rise to separate transmission chains that still exist today. We may never know how many individuals were infected in the previous transmission chain, the one that led from the person initially infected with SIV to the progenitor of the current pandemic in humans." Paul Sharp & Beatrice Hahn (see below)
News
Tissue sample suggests HIV has been infecting humans for a century
Published online 1 October 2008 | Nature | doi:10.1038/news.2008.1143
48-year-old lymph node biopsy reveals the history of the deadly virus.
Heidi Ledford
A biopsy taken from an African woman nearly 50 years ago contains traces of the HIV genome, researchers have found. Analysis of sequences from the newly discovered sample suggests that the virus has been plaguing humans for almost a century.
Although AIDS was not recognized until the 1980s, HIV was infecting humans well before then. Researchers hope that by studying the origin and evolution of HIV, they can learn more about how the virus made the leap from chimpanzees to humans, and work out how best to design a vaccine to fight it.
In 1998, researchers reported the isolation of HIV-1 sequences from a blood sample taken in 1959 from a Bantu male living in Leopoldville1 - now Kinshasa, the capital of the Democratic Republic of the Congo. Analysis of that sample and others suggested that HIV-1 originates from sometime between 1915 and 19412.
Now, researchers report in Nature that they have uncovered another historic sample, collected in 1960 from a woman who also lived in Leopoldville3.
"It's as if you had a nice pearl necklace of DNA and RNA and protein and you clumped it together, drenched it in glue and then dried it out."
Michael Worobey
University of Arizona in Tucson
It took evolutionary biologist Michael Worobey of the University of Arizona in Tucson and his colleagues eight years of searching for suitable tissue collections originating in Africa before they tracked down the 1960 lymph node biopsy at the University of Kinshasa.
Drenched in glue
The samples had all been treated with harsh chemicals, embedded in paraffin wax and left at room temperature for decades. The acidic chemicals had broken the genome up into small fragments. Formalin, a chemical used to prepare samples for microscopy, had crosslinked nucleic acids with protein. "It's as if you had a nice pearl necklace of DNA and RNA and protein and you clumped it together, drenched it in glue and then dried it out," says Worobey.
The team worked out a combination of methods that would allow them to sequence DNA and RNA from the samples; another lab at Northwestern University in Chicago, Illinois, confirmed the results, also finding traces of the HIV-1 genome in the lymph node biopsy.
Kinshasa was founded around 1885. The growth of Kinshasa and other cities in the region may have been crucial to the emergence of HIV/AIDS.Royal Museum for Central Africa
Using a database of HIV-1 sequences and an estimate of the rate at which these sequences change over time, the researchers modelled when HIV-1 first surfaced. Their results showed that the most likely date for HIV's emergence was about 1908, when Leopoldville was emerging as a centre for trade.
Although that date will not surprise most HIV researchers, the new data should help persuade those who were unconvinced by the 1959 sample, says Beatrice Hahn, an HIV researcher at the University of Alabama at Birmingham.
The sequences of the 1959 and 1960 samples - the earliest that have ever been found - show a difference of about 12%. "This shows very clearly that there was tremendous variation even then," says Simon Wain-Hobson, a virologist at the Pasteur Institute in Paris.
A virus ready for its close-up
However, it may never be possible to pinpoint exactly how HIV crossed from chimpanzees into humans, Hahn cautions. She and her collaborators previously tracked the likely source of HIV-1 to chimpanzees living in southeast Cameroon4, hundreds of kilometres from Kinshasa, and it is tempting to hypothesize that trade routes contributed to the virus's infiltration of the city. But even by 1960, HIV-1 had infected only a few thousand Africans. It is unlikely that it will be possible to track down samples from the very earliest victims, Hahn notes.
Meanwhile, Worobey plans to continue his search through old tissue collections in the hope of finding additional samples. In time, he says, it may even be possible to reconstruct the historic HIV viruses for further study.
Collecting information about old strains of HIV - even those that disappeared over time - can help researchers learn how successful strains broke through, says Wain-Hobson. "For every star in Hollywood there are fifty starlets," he says. "We would love to know what it was that caused this strain to move out of starlet phase and to the big time."
News and Views
Nature 455, 605-606 (2 October 2008)
AIDS: Prehistory of HIV-1
Paul M. Sharp1 & Beatrice H. Hahn2
The origin of the current AIDS pandemic has been a subject of great interest and speculation. Viral archaeology sheds light on the geography and timescale of the early diversification of HIV-1 in humans.
Human immunodeficiency virus type 1 (HIV-1) must have been spreading through the human population long before AIDS was first described in 1981, but very few strains from this 'prehistoric' period (pre-1980s) have been characterized. Viral sequences from earlier times can provide insight into the early spread of HIV-1, because the rapid rate of evolution of this virus - up to a million times faster than that of animal DNA - means that substantial amounts of sequence change occur in a matter of decades1. On page 661 of this issue, Worobey et al.2 describe the sequences of partial genome fragments of HIV-1 from a lymph-node biopsy collected in 1960 in Leopoldville (now Kinshasa, Democratic Republic of the Congo). They compare these sequences with those of other HIV-1 strains, shedding light on the early evolution and diversification of this virus in Africa.
HIV-1 strains are divided into three groups, each of which was independently derived from a simian immunodeficiency virus (SIV) that naturally infects chimpanzees in west-central Africa3. Whereas two of these groups are rare, the third, group M, has spread throughout the world and is the cause of more than 95% of HIV infections globally. Group M can be further divided into many subtypes (A-K), which seem to have arisen through founder events. For example, subtype B, which encompasses all the strains originally described in North America and Europe, is very rare in Africa, and reflects such a founder event. Last year, Worobey and colleagues showed4 that this subtype probably arose from a single strain that was carried from Africa to Haiti before spreading to the United States and onwards. The newly described2 1960 virus (DRC60) falls within, but close to the ancestor of, subtype A.
DRC60 is not the first 'ancient' HIV-1 sample to be characterized: viral sequences from a blood-plasma sample originally obtained in 1959 - also from Leopoldville - were published 10 years ago5. The importance of DRC60 is that it is highly divergent from the 1959 sample (ZR59), which was most closely related to the ancestor of subtype D, thus directly demonstrating that, by 50 years ago, group M HIV-1 strains had already undergone substantial diversification.
The ZR59 and DRC60 sequences differ by about 12%, a value similar to distances now seen between the most divergent strains within subtypes. As the positions of ZR59 and DRC60 within the group M phylogeny indicate that the various subtypes already existed 50 years ago, simple extrapolation suggests that these two viral sequences had a common ancestor at least 50 years before that. For a more robust estimate of the date of the common ancestor of HIV-1 group M strains, Worobey and colleagues used state-of-the-art statistical analyses, allowing a variety of models for the growth of the HIV-1 pandemic and variable rates of evolution. The different analyses gave broadly similar estimates for the date of that common ancestor, between 1902 and 1921, with 95% confidence intervals ranging no later than 1933. These dates are a little earlier than, but do not differ significantly from, a previous estimate1 of 1931 from an analysis that did not include the 50-year-old viruses.
The interpretation that HIV-1 was spreading among humans for 60-80 years before AIDS was first recognized should not be surprising. If the epidemic grew roughly exponentially from only one or a few infected individuals around 1910 to the more than 55 million estimated to have been infected by 2007, there were probably only a few thousand HIV-infected individuals by 1960, all in central Africa. Given the diverse array of symptoms characteristic of AIDS, and the often-long asymptomatic period following infection, it is easy to imagine how the nascent epidemic went unrecognized. Conversely, such a low prevalence at that time implies that the Congolese co-authors of the paper2 were very lucky to come across this infected sample, even if most infections were concentrated in the area of Leopoldville. But can we trust these sequences?
In work on ancient DNA, contamination is especially problematic, and the work should, if possible, be replicated in other laboratories. For DRC60, independent analyses were performed at the University of Arizona and Northwestern University, Illinois. The sequences obtained were similar, but not identical, exactly as expected when samples come from the diverse set of related viral sequences that - because of the virus's rapid rate of evolution - arise within an infected individual6. Furthermore, the distance along the evolutionary tree from the group M ancestor to the ZR59 or DRC60 sequences is much shorter than those between the ancestor and modern strains, consistent with the earlier dates of isolation of ZR59 and DRC60, and confirming that these viruses are indeed old.
Although the ZR59 and DRC60 sequences can show only that two subtypes were present in Leopoldville around 1960, in more recent times the greatest diversity of group M subtypes - as well as many divergent strains that have not been classified - has been found in Kinshasa7. So it seems likely that all of the early diversification of HIV-1 group M viruses occurred in the Leopoldville area. Yet the SIV strains most closely related to HIV-1 group M have been found infecting chimpanzees in the southeast corner of Cameroon3, some 700 kilometres away (Fig. 1a). The simplest explanation for how SIV jumped to humans would be through exposure of humans to the blood of chimpanzees butchered locally for bushmeat. So why did the pandemic start in Leopoldville? And, as there must have been many opportunities for such transmission over past millennia, why did the AIDS pandemic not occur until the twentieth century?
Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960
Letter
Nature 455, 661-664 (2 October 2008) | doi:10.1038/nature07390; Received 21 May 2008; Accepted 8 September 2008
Michael Worobey1, Marlea Gemmel1, Dirk E. Teuwen2,3, Tamara Haselkorn1, Kevin Kunstman4, Michael Bunce5, Jean-Jacques Muyembe6,7, Jean-Marie M. Kabongo6, Raphal M. Kalengayi6, Eric Van Marck8, M. Thomas P. Gilbert1,9 & Steven M. Wolinsky4
1. Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA
2. Sanofi Pasteur, F-69367 Lyon Cedex 07, France
3. UCB SA Pharma, Braine l'Alleud, BE-1420, Belgium
4. The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
5. Ancient DNA Laboratory, School of Biological Sciences and Biotechnology, Murdoch University, Perth, Western Australia 6150, Australia
6. Department of Anatomy and Pathology, University of Kinshasa, Kinshasa B.P. 864, Democratic Republic of the Congo
7. National Institute for Biomedical Research, National Laboratory of Public Health, Kinshasa B.P. 1197, Democratic Republic of the Congo
8. Department of Pathology, University Hospital, University of Antwerp, Antwerp B-2610, Belgium
9. Present address: Centre for Ancient Genetics, Biological Institute, University of Copenhagen, Copenhagen DK-2100, Denmark.
Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution1, 2. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection1. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Leopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods.
We screened 27 tissue blocks (8 lymph node, 9 liver and 10 placenta) obtained from Kinshasa between 1958 and 1960 by polymerase chain reaction with reverse transcription (RT-PCR); one lymph node biopsy specimen contained HIV-1 RNA. Viral nucleic acids were extracted from this specimen using protocols optimized for the recovery of nucleic acids from ancient or degraded samples3, 4. After reverse transcription, 12 out of the 14 short HIV-1 complementary DNA fragments in the study (Fig. 1a) were amplified by PCR using a panel of conserved primer pairs from different regions of the viral genome (Supplementary Table 1). Each PCR product was cloned and sequenced. Sequences were reproducible after repeated extractions and were not the result of PCR contamination (see Fig. 1a and Supplementary Table 1 for fragment designations). The results were confirmed independently in two laboratories (Fig. 1b and Supplementary Fig. 1), with the second laboratory successfully identifying the positive 1960 specimen in a blinded assay. The short fragments of the 1960 sample were found to be of subtype A and not to be a mosaic of contemporary sequences (see Supplementary Information for a detailed discussion of the authenticity of the 1960 sequences). Consensus nucleotide sequences from these short HIV-1 fragments were concatenated for study. The analyses included reference sequences from the Los Alamos National Laboratory HIV sequence database and sequences recovered as part of this study from three paraffin-embedded tissue specimens collected from AIDS patients in Belgium and Canada between 1981 and 1997.
HIV-1 sequences were analysed in MrBayes v3.1.2 (ref. 5) using an unconstrained (in which a molecular clock was not enforced) Bayesian Markov chain Monte Carlo (BMCMC) method. The phylogenetic analyses confirmed that the DRC60 consensus sequences from the two laboratories were derived from a single patient (uncorrected pairwise distance of 1.4%). The sequences were positioned close to the ancestral node of the subtype A lineage (excluding sub-subtype A2), forming a monophyletic clade with three modern sequences from the DRC (Fig. 1b and Supplementary Fig. 1). Assuming a similar rate of evolution along all branches on a tree, the divergence between two sequences reflects the time elapsed since their shared ancestor. As predicted, the DRC60 sequences had a shorter branch length to the A/A1 ancestral node than the contemporary subtype A viruses sampled from the same geographic region (P = 1.0).
We validated the time of origin of the 1960 sequence by comparisons of the predicted date to the documented date. With the DRC60 date treated as an unknown, we calculated an evolutionary rate on the basis of the distribution of branch lengths on the unconstrained phylogenetic trees sampled by MrBayes. To limit the effects of evolutionary rate differences between clades and uncertainties in rooting the HIV-1 M group phylogeny, we focused on the subtype A/A1 subtree (Supplementary Fig. 1) and analysed root-to-tip branch lengths relative to the sampling year. The mean estimates for the year of origin of the DRC60 consensus sequences from the University of Arizona and Northwestern University laboratories were 1959 (95% highest probability distribution (HPD) 1902-1984) and 1959 (95% HPD 1915-1985), respectively, corroborating the authenticity of the DRC60 sequences and the existence of a clock-like signal in our data set (see later). Despite initial indications that recombination might seriously confound phylogenetic dating estimates6, subsequent work has suggested that recombination is not likely to systematically bias HIV-1 dates in one direction or the other, although it is expected to increase variance7. The close match between the predicted and the actual dates of both ZR59 (ref. 2) and DRC60 provides support for this view and gives an unambiguous indication that HIV-1 evolves in a fairly reliable clock-like fashion.
The uncorrected pairwise distance between DCR60 and ZR59 in their overlapping env region was 11.7% (Fig. 1c). This genetic distance is greater than 99.2% of within-subtype comparisons (within-subtype difference, range 0.01-0.15; between-subtype difference, range 0.05-0.18). Because each subtype represents several decades of independent evolution in the human population2, 8, the extensive divergence between DRC60 and ZR59 indicates that the HIV-1 M group founder virus began to diversify in the human population (and that HIV-1 probably entered Kinshasa) decades before 1960.
We applied a relaxed clock BMCMC coalescent framework as implemented in BEAST v1.4.7 (ref. 9) to estimate the time to the most recent common ancestor (TMRCA) of the HIV-1 M group. This approach robustly incorporates phylogenetic uncertainty and accounts for the possibility of variable substitution rates among lineages and differences in the demographic history of the virus, sampling phylogenies and parameter estimates in proportion to their posterior probability10. As with other studies of HIV-1 (ref. 11), comparisons of the marginal likelihoods of strict versus relaxed clock models (both of which are implemented in BEAST) indicated overwhelming support for relaxed clocks (data available on request). Hence, the use of strict clock models with these data would be inappropriate and would probably yield misleadingly small error estimates with regard to both timing and substitution rates.
Using substitution rates calibrated with sequences sampled at different time points, we obtained a posterior distribution of rooted tree topologies with branch lengths in unit time (Fig. 2 and Supplementary Fig. 2). The median estimated substitution rate for the concatenated subregions of the gag-pol-env genes was 2.47 10-3 substitutions per site per year (95% HPD 1.90-2.95 10-3). The inclusion of the 1959 and 1960 sequences seemed to improve estimation of the TMRCA of the M group (Table 1), limiting the influence of the coalescent tree prior on the posterior TMRCA distributions compared with the data set that excluded these earliest cases of HIV-1. With DRC60 and ZR59 included, the different demographic/coalescent models gave highly consistent results, with tighter and more similar date ranges compared with the analyses that excluded them and 95% HPDs that extend no later than 1933. The best-fit model incorporated a constant population size demographic model (TMRCA 1921, 95% HPD 1908-1933). The model with a general, non-parametric prior (the Bayesian skyline plot tree prior)12, 13 that indicated a more complex (and biologically plausible) demographic history (Supplementary Fig. 3) had a statistically indistinguishable degree of support (TMRCA 1908, 95% HPD 1884-1924). Moreover, the population expansion demographic model9, which was a slightly worse fit to the data compared with the constant population and Bayesian skyline plot models, could not be rejected given the Bayes factor comparison of models (Table 1). The inability to strongly reject the model with a constant population size prior is counterintuitive because it is clear that the HIV-1 population size has increased notably. We speculate that this finding might be due to the simplest model providing a good fit to a relatively short, information-poor alignment, in comparison with more parameterized models.
Acid-containing fixatives such as Bouin's solution can cause base modifications of nucleic acids, leading to the generation of erroneous bases in sequences derived from such samples3. However, the replication of all sequences from independent PCR amplifications and the uncorrected pairwise distance between the consensus sequences from the two laboratories (1.4%) suggest that few of the mutations on the DRC60 lineage are damaged-induced. Moreover, our relaxed clock methods are likely to be fairly robust to the presence of such mutations in one lineage9. Nevertheless, additional old sequence data would be helpful for resolving what impact, if any, this possible source of error had on the slightly earlier dates we calculated compared with previous estimates that did not include early calibration points2, 8, 14, 15. Interestingly, the best-fit model for the data set that excluded ZR59 and DRC60 (Table 1) gave a TMRCA estimate of 1933 (1919-1945), which is very similar to that of ref. 2. This suggests that the inclusion of the old sequences, rather than the vagaries associated with a much shorter alignment than that analysed by ref. 2, might explain the discrepancy. Also, one earlier study, using sequences from the DRC only16, produced dating and demography estimates very similar to ours. Overall, there is broad agreement between all of these studies in spite of differences in data and methods.
Our estimation of divergence times, with an evolutionary timescale spanning several decades, together with the extensive genetic distance between DRC60 and ZR59 indicate that these viruses evolved from a common ancestor circulating in the African population near the beginning of the twentieth century; TMRCA dates later than the 1930s are strongly rejected by our statistical analyses. The topology of the HIV-1 group M phylogeny provides further support for this conclusion. Unlike ZR59, which is basal to subtype D1, DRC60 branches off from the ancestral node of subtype A/A1 (Fig. 2 and Supplementary Figs 1 and 2). Thus, it is clear that phylogenetically distinct subtypes (and/or their progenitors) were already present in the DRC by this early time point (Fig. 2). Notably, DRC60 and ZR59 cluster with other strains from the same geographical region and basal to other members of their respective subtypes, a pattern consistent with the hypothesis that the subtypes spread through lineage founder effects worldwide, whereas a more diverse array of forms remained at the site of origin in Africa17, 18.
The reservoir of the ancestral virus still exists among wild chimpanzee communities in the same area on the African continent19. Humans acquired a common ancestor of the HIV-1 M group by cross-species transmission under natural circumstances20, probably predation21. The Bayesian skyline plot (Supplementary Fig. 2), which tracks effective population size through time, suggests that HIV-1 group M experienced an extensive period of relatively slow growth in the first half of the twentieth century. A similar pattern has been inferred using sequences sampled only in the DRC16. This pattern, and the short duration between the first presence of urban agglomerations in this area and the timing of the most recent common ancestor of HIV-1 group M (Fig. 3), suggests that the rise of cities may have facilitated the initial establishment and the early spread of HIV-1. Hence, the founding and growth of colonial administrative and trading centres such as Kinshasa22 may have enabled the region to become the epicentre of the HIV/AIDS pandemic23.
The archival banks of Bouin's-fixed paraffin-embedded tissue specimens accumulated by many hospitals in west-central Africa provide a vast source of clinical material for viral genetic analysis. As with the 1918 Spanish influenza pandemic virus24, 25, a deep perspective on the evolutionary history of HIV-1 using sequences resurrected from the earliest cases in Africa could yield important insights into the pathogenesis, virulence and evolution of pandemic AIDS viruses.
Methods Summary
A total of 813 Bouin's-fixed paraffin-embedded histopathological blocks were recovered from the 1958-1962 archives of the Department of Anatomy and Pathology at the University of Kinshasa. The boxes were stored until transfer to the University of Arizona, where 8 lymph node, 9 liver and 10 placenta samples from 1958-1960 were selected for RNA preservation analysis and HIV-1 RNA screening. We used a human -2-microglobulin (B2M) quantitative RT-PCR assay to assess RNA quality as described3. Digestion and extraction of these samples, and of three modern positive-control samples, were performed using QIAamp DNA micro kits (Qiagen) using the protocol described in ref. 3. We used 14 primer sets designed to anneal to highly conserved regions of the gag, pol and env genes of HIV-1 group M and to amplify very short fragments likely to be present even in ancient and/or degraded specimens (Supplementary Table 1). Reverse transcription was performed using the SuperScript III System for RT-PCR (Invitrogen). The cDNA was amplified by PCR using Platinum Taq HiFi enzyme (Invitrogen) and cloned using the TOPO TA Cloning Kit (Invitrogen). We constructed an alignment including 156 published reference sequences plus the sequences recovered in this study, concatenating the 12 (out of 14) fragments successfully amplified from the 1960 sample and the 4 fragments already available from the 1959 sample (994 bases total). We performed an unconstrained (not enforced by a molecular clock) BMCMC analysis in MrBayes v3.1.2 (ref. 5) and used the resulting MCMC sample to test whether the 1960 sequence exhibited properties consistent with its provenance (both age and geography). We used a relaxed molecular clock model, as implemented in BEAST v1.4.7 (ref. 9), to estimate the TMRCA of HIV-1 group M using the 1960 and 1959 samples and to investigate the demographic history of the virus. We also performed pairwise comparisons within and between subtypes for the 163 bases available for both DRC60 and ZR59.
|
|
| |
| |
|
|
|
