Rafael Nájera

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance-a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Molecular epidemiology of HIV-1 genetic forms and its significance for vaccine development and therapy

Since their initial expansion in human beings roughly seven decades ago in central Africa, the HIV-1 pandemic strains have diversified extensively through mutation and recombination. 24 circulating genetic forms of the main HIV-1 group are presently recognised, including 11 subtypes or sub-subtypes and 13 circulating recombinant forms. New genetic forms are being introduced in different areas of the world, changing the molecular epidemiology of the infection. It is generally agreed that the control of the HIV-1 pandemic requires the development of vaccines that efficiently protect against the range of HIV-1 genetic forms. The introduction of effective antiretroviral therapies in areas of high HIV-1 prevalence may also contribute to the control of the pandemic, as has been documented in developed countries. Efficient targeting of the extensive genetic diversity of HIV-1 constitutes one of the major challenges in present efforts against the pandemic, although the significance of HIV-1 genetic forms for vaccine development and therapy remains to be defined.

Travel and the Introduction of Human Immunodeficiency Virus Type 1 Non-B Subtype Genetic Forms into Western Countries

Both high mutation rates and recombination contribute to the genetic diversity of human immunodeficiency virus type 1 (HIV-1). Among viruses of the main group, which are responsible for the HIV-1 pandemic, 21 circulating genetic forms have been reported, 11 of which are recombinant between > or = 2 subtypes. In Western Europe and the Americas, the HIV-1 epidemic is largely dominated by B subtype viruses; however, infections with diverse non-B subtype genetic forms are increasingly being recognized. In Western Europe and North America, most of them have been identified in immigrants or travelers returning from areas with high HIV-1 prevalence, mainly from sub-Saharan Africa and Southeast Asia, where non-B subtype genetic forms predominate, but propagation within other groups has been reported in some Western countries. This may have implications for prophylactic and therapeutic strategies and, by bringing in contact different genetic forms, may favor the generation of novel recombinant viruses. Travelers from different categories--including immigrants, military personnel, seamen, tourists, expatriates, diplomats, and businessmen--may be at risk of transporting HIV non-B subtype genetic forms to Western countries.

Identification of a newly characterized HIV-1 BG intersubtype circulating recombinant form in Galicia, Spain, which exhibits a pseudotype-like virion structure

Summary: We recently reported the finding of phylogenetically related HIV‐1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full‐length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV‐1 circulating recombinant form (CRFI4_BG), the first reported to originate in Western Europe.

HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users.

BackgroundThe HIV-1 epidemics in Western Europe are dominated by B subtype viruses. Non-B subtype is largely restricted to individuals infected outside of Europe and to their direct contacts and is generally acquired by the heterosexual route. MethodsProtease and a segment of reverse transcriptase were amplified and sequenced from plasma RNA in 451 individuals from seven cities of Galicia, north-western Spain. Subtype sequence homologies were determined using the BLAST algorithm. Non-B sequences were examined by phylogenetic analysis and intersubtype recombination by bootscanning. The env V3 region was analysed in all non-B and in 38 B subtype viruses. ResultsTen different non-B genetic forms were identified in 20 (4.4%) individuals. Subtypes were concordant between pol and V3 in five viruses; 14 (70%) infections were with intersubtype recombinant viruses, and one individual had a dual B+G infection. Seven recombinant viruses were phylogenetically related to five reported recombinant forms. Three non-recombinant G and six recombinant BG viruses formed a monophyletic cluster for pol. All but three individuals with non-B infections were native Spanish. Only 6 of 16 individuals referred to sexual contacts with sub-Saharan Africans. Twelve (60%) non-B subtype infections, including all with G and BG viruses, were in injecting drug users (IDU). ConclusionsNon-B subtype viruses were identified in 4.4%, with a high diversity of genetic forms, including 70% infections with intersubtype recombinant viruses. The majority of individuals with non-B infections were IDU, most of them without known contacts with non-European sources, and among whom BG recombinant viruses are circulating.

Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome

The genetic heterogeneity of five influenza A (H3N2) strains isolated between 1968 and 1977 has been estimated by T1-oligonucleotide fingerprinting of 32P-labeled viral RNA. Assuming that the large T1-resistant oligonucleotides represent a random sample of the viral RNA, the genetic differences observed would affect 0.3 to 10.7% of the RNA positions of the genes studied, depending on the pair of viruses considered. A smaller degree of genetic heterogeneity was observed when six coetaneous viral samples were compared. The distribution of spontaneous mutations among the viral genes was studied by fingerprinting individual RNA segments isolated either by gel electrophoresis or hybridization with plasmids containing influenza-specific DNA sequences. No statistically significant differences were detected in the distribution of mutations among the viral genes studied. The mutation frequency at the hemagglutinin RNA region coding for the HA1 subunit was found to be two times higher than that at the region encoding that HA2 subunit. Our results suggest that the antigenic variability of influenza viruses may be a consequence of a general genetic variability which effects many of the viral genes.

High HIV-1 genetic diversity in Cuba.

BackgroundHIV-1 subtype B is largely predominant in the Caribbean, although other subtypes have been recently identified in Cuba. ObjectivesTo examine HIV-1 genetic diversity in Cuba. MethodsThe study enrolled 105 HIV-1-infected individuals, 93 of whom had acquired the infection in Cuba. DNA from peripheral blood mononuclear cells was used for polymerase chain reaction amplification and sequencing of pol (protease–reverse transcriptase) and env (V3 region) segments. Phylogenetic trees were constructed using the neighbour-joining method. Intersubtype recombination was analysed by bootscanning. ResultsOf the samples, 50 (48%) were of subtype B and 55 (52%) of diverse non-B subtypes and recombinant forms. Among non-B viruses, 12 were non-recombinant, belonging to six subtypes (C, D, F1, G, H and J), the most frequent of which was subtype G (n = 5). The remaining 43 (78%) non-B viruses were recombinant, with 14 different forms, the two most common of which were Dpol/Aenv (n = 21) and U(unknown)pol/Henv (n = 7), which grouped in respective monophyletic clusters. Twelve recombinant viruses were mosaics of different genetic forms circulating in Cuba. Overall, 21 genetic forms were identified, with all known HIV-1 group M subtypes present in Cuba, either as non-recombinant viruses or as segments of recombinant forms. Non-B subtype viruses were predominant among heterosexuals (72%) and B subtype viruses among homo- or bisexuals (63%). ConclusionAn extraordinarily high diversity of HIV-1 genetic forms, unparalleled in the Americas and comparable to that found in Central Africa, is present in Cuba.

Identification of a novel HIV-1 complex circulating recombinant form (CRF18_cpx) of Central African origin in Cuba.

Background:Analysis of partial pol and env sequences have indicated a high diversity of HIV-1 genetic forms in Cuba, including two potential novel circulating recombinant forms (CRF): Upol/Henv and Dpol/Aenv. Objectives:To determine whether Upol/Henv recombinant viruses from Cuba, detected in 7% of samples, represent a novel HIV-1 CRF, and to identify non-Cuban viruses related to this recombinant form. Methods:Near full-length genome amplification was carried out by nested polymerase chain reaction in four overlapping DNA segments of two epidemiologically unlinked viruses in uncultured peripheral blood mononuclear cells. The sequences were analysed phylogenetically. Recombinant structures and phylogenetic relationships were analysed by bootscanning and by maximum likelihood. Searches for related viruses in databases were initially based on sequence homology and sharing of signature nucleotides. Results:Both Cuban viruses clustered uniformly in bootscans all along the genome with each other and with a virus from Cameroon, CM53379, indicating that all three represent the same recombinant form. Their genome comprised multiple segments clustering with subtypes A1, F, G, H and K, as well as segments failing to cluster with recognized subtypes. The newly defined CRF, designated CRF18_cpx, was phylogenetically related in partial segments to CRF13_cpx, CRF04_cpx and 36 additional viruses, most of them from Central Africa. One of the viruses from Cameroon, sequenced in the near full-length genome, was a CRF18_cpx/subtype G secondary recombinant. Conclusions:A novel HIV-1 complex circulating recombinant form (CRF18_cpx) has been identified that is circulating in Cuba and Central Africa.

Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19_cpx) in Cuba.

Circulating recombinant forms (CRFs) represent a substantial proportion of HIV-1 isolates in the global pandemic. Characterization of HIV-1 genetic forms, including CRFs, may be relevant to studies on molecular epidemiology, recombination, superinfection, vaccine development, and antiretroviral therapy. This study analyzes near complete genomes of 4 epidemiologically unlinked viruses from Cuba, originally characterized as D/A intersubtype recombinants in pol and env segments. The genomes of 3 viruses exhibited virtually coincident mosaic structures, with multiple segments of subtypes A, D, and G and uniform phylogenetic clustering with each other along the genome. These results allow us to define a new CRF (CRF19_cpx). The 4th analyzed Cuban virus was recombinant between CRF19_cpx and CRF18_cpx (which also circulates in Cuba). CRF19_cpx exhibited homology to an AG intersubtype recombinant virus from Cameroon (CM53392) along approximately 5 kb and clustered with a subtype D virus from Gabon (G109) in gag. Four other viruses from central or west Africa were also phylogenetically related to CRF19_cpx in env fragments. These results allow us to define CRF19_cpx as a second novel CRF of African origin circulating in Cuba, to identify putative representative viruses of its parental strains, and to characterize a unique CRF18/CRF19 recombinant virus.