Human T cell lymphotropic viruses types 1 and 2 (HTLV-1 and -2) - Antibodies; Antibodies confirmation test; Molecular diagnosis (RT-PCR); Proviral DNA (PCR)


Information 27-05-2018.


The human T-cell lymphotropic viruses (HTLV) belong to the family Retroviridae, subfamily Oncovirinae, genus Deltaretrovirus. The first member, the human T-cell leukemia virus type 1 (HTLV-1), was the first human retrovirus discovered in 1980 and was identified as the etiological agent of a T-cell leukemia in adults, and subsequently, it was associated with tropical spastic paraparesis and other processes. Shortly thereafter, another human retrovirus was discovered, the human T cell leukemia virus type 2 (HTLV-2) very similar to HTLV-1 in the structure of the genome and the nucleotide sequence. In 2005, a third and fourth type of HTLV closely related to HTLV-1, human T cell leukemia virus type 3 (HTLV-3) and human T cell leukemia virus type 4 (HTLV-4), were discovered in central Africa and are currently being studied.

The HTLV-1 and HTLV-2 viruses show considerable homology in terms of genome structure, replication pattern, and properties of structural, regulatory and accessory proteins. Despite these important analogies, HTLV-1 and HTLV-2 are markedly different in terms of clinical impact, and only HTLV-1 is conclusively associated with neoplasms, which develop in up to 5% of infected individuals. The HTLV-1 virus is the etiological agent of adult T cell leukemia/lymphoma and tropical spastic paraparesis or HTLV-1 associated myelopathy. It is also associated with the development of other clinical entities such as chronic inflammatory arthropathy, Sjögren's syndrome, polymyositis, uveitis, alveolitis, strongyloidiasis and infectious dermatitis. Despite its involvement in human pathology, it is important to note that more than 80% of individuals seropositive for HTLV-1 do not develop any clinical manifestation throughout their life and that the small percentage that does, develops these diseases after a prolonged viral persistence, usually after two decades of infection. Unlike HTLV-1, HTLV-2 has not been convincingly associated with human pathology. This virus is persistently associated with elevated lymphocyte and platelet counts and an increase in overall mortality from cancer, but does not cause hematological disorders and is only sporadically associated with myelopathy. Although its definitive role in human pathology is still not well defined, certain neurological, dermatological and hematological processes linked to HTLV-2 infection have been described.

Both HTLV-1 and HTLV-2 have a worldwide distribution and are included in prevalence estimates worldwide. It is estimated that there are between 10-20 million people infected with HTLV-1 worldwide, mostly in Japan and the Caribbean Basin. The distribution of HTLV-1/2 is heterogeneous, with areas of high endemicity and other areas where these retroviruses have little or no presence. HTLV-1 is endemic in Japan, sub-Saharan Africa, South America and the Caribbean. While HTLV-2 is endemic among aborigins in Latin America and some tribes in Africa, it prevails among drug users in Europe and North America.

The structure of the HTLV-1 and -2 viruses consists of a lipid-protein envelope and a central nucleocapsid called "core", where the genetic material and certain enzymes necessary for its life cycle are located, including reverse transcriptase. The genetic material of HTLV-1/2 retroviruses consists of two identical molecules of single-stranded RNA and positive polarity. The genome of HTLV-1 consists of 9,032 nucleotides and that of HTLV-2 of 8,932. It is composed of the gag, pol and env genes, which encode internal proteins, enzymes and envelope proteins, respectively. In addition, the genome of these viruses includes a region called "X", which codes for proteins that regulate viral replication, including Tax, Rex and HBZ. This "X" region is separated from the main fragment, which codes for the rest of proteins, by a silent region, not translated into proteins. In addition, the genomic RNA is flanked on both sides by long terminal repeat sequences (LTRs), which have an important role in the integration of proviral DNA of HTLV-1/2 in the genome of the cell they infect.

HTLVs produce a persistent and slow infection in the host that they infect, and they are transmitted vertically and horizontally through the transfer of infected lymphocytes through perinatal transmission, breastfeeding, sexual contact, blood transfusion and intravenous drug use. The infection begins with the adsorption of the virus, which occurs through cell surface receptors that recognize the glycoproteins of the viral envelope, mainly gp46. Both viruses use the cellular receptors GLUT-1 and neuropilin-1 (NRP1) for their entry into the host cell, although HTLV-1, but not HTLV-2, depends on the proteoglycans of heparan sulfate. Although the use of receptors allows HTLV-1 and HTLV-2 to infect different cell types in vitro, in vivo they exhibit distinctive cell tropisms: HTLV-1 mainly infects CD4 + T cells, while CD8 T cells + are the preferred target of HTLV-2. However, they can also be detected in other cell types, such as dendritic cells, monocytes, macrophages, fibroblasts and B lymphocytes.

Once inside the host cell, one of the RNA strands of the viruses is transcribed to DNA by reverse transcriptase. This complementary DNA is integrated, in the form of a provirus, in the genome of the host cell, of the T lymphocyte. After being integrated as a provirus into the cellular genome, the HTLVs multiply mainly by clonal expansion of the host cell, starting from mitosis of the cells that it infects. In this way, clonal expansion occurs using cellular enzymes, DNA polymerases with error correction capability. In addition, cell-to-cell transmission also occurs through the formation of a viral synapse, which involves a cell-cell contact, with polarization of the microtubule-organizing center and directional release of virions from the infected to the uninfected cell. Therefore, unlike HIV, which has an important genomic variability, HTLVs are relatively stable. The low genetic variability is mainly due to the absence or low frequency of replicative cycles using viral reverse transcriptase, known to introduce high frequency mutations. This characteristic determines that the infectivity associated with the free extracellular particles is very low and promotes the persistence of the infection in the organism by evading the immune response of the host. The studies carried out indicate that at the beginning of the infection with HTLV-1 there is an active replication phase, where both the mechanism of clonal expansion via mitosis and the replication via reverse transcriptase are actively involved in the progression of the infection. However, the host develops an immune response directed against the cells in which the virus is being transcribed and which produce viral proteins. In particular, the main target appears to be the product of the gene tax, which is expressed on the cell surface of infected lymphocytes within the context of the HLA system. That is why a Tax / Rex / HBZ regulatory system has been postulated in which Tax activates HBZ and, at the same time, this protein, represses the regulation of transcription mechanisms mediated by Tax / Rex. Therefore, during HTLV-1 infection, when Tax / Rex expression is robust and dominant over HBZ, productive infection occurs with expression of structural proteins and the hyperactivation of NF-κB, which induces senescence of the cells However, when the expression of Tax / Rex is silenced and HBZ is dominant, latent infection is established with expression of regulatory proteins (Tax / Rex / HBZ), but not structural. Thus, HBZ maintains viral latency by downregulating the activation of senescence induced by Tax, and by inhibiting Rex-mediated expression of viral structural proteins. This silencing would be a form of escape to the immune response by the infected cells, which means that the virus can only persist in a silent, proviral form and be perpetuated by cellular mitosis. The regulation and interaction of Tax / HBZ proteins is also an important factor in the development of oncogenesis of HTLV-1. Both proteins can promote the proliferation and immortalization of infected T cells. In fact, it is thought that the differences between the regulatory proteins Tax1 and Tax2, encoded by HTLV-1 and -2, respectively, may be responsible for the different pathogenicities of retroviruses. The Tax2 regulator lacks the motif of binding to the PDZ domain (PBM) present in Tax1 and important for cell transformation activity.

The diagnosis of HTLV infection can be made by direct or indirect methods. Indirect methods are the most widely used and are based on the recognition of specific antibodies against virus proteins, serum or other biological fluids. The most common techniques to detect anti-HTLV antibodies are enzyme-linked immunosorbent assays (EIA). However, these methods are unable to discriminate if the infection is caused by HTLV-1 or HTLV-2. For this reason, all positive results must be confirmed by more specific techniques, such as western blot (WB), indirect immunofluorescence or radioimmunoprecipitation, which are capable of recognizing each of the antibodies directed against the different proteins of both viruses. On the other hand, the methods that demonstrate the presence of the virus directly are the viral culture and the polymerase chain reaction (PCR). PCR is a molecular biology technique of great sensitivity and specificity that allows detecting not only the presence of extracellular particles of HTLVs by detecting the RNA of the virus, indicating that the virus is in the active replication phase, but also, the detection of the virus integrated in the lymphocytes by means of the detection of the viral complementary DNA. Due to the minimal or absent extracellular phase of HTLV-1/2 in vivo, by not using its reverse transcriptase in its expansion phase in the infected subject, the detection of the proviral DNA of these viruses is an important tool for determining the infection. Cases of positive PCR for HTLV-1 and HTLV-2 have been reported in the absence of seropositivity, or with undetermined patterns in WB, particularly in immunosuppressed subjects coinfected with HIV, and in patients with neurological diseases. Therefore, in indeterminate cases or HTLV without typing by WB, it is recommended to use a nested polymerase chain reaction to confirm the infection.

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