Retroviral Research Articles

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target.

L-type amino acid transporter 1 (LAT1)/SLC7A5 is the first identified CD98 light chain disulfide linked to the CD98 heavy chain (CD98hc/SLC3A2). LAT1 transports large neutral amino acids, including leucine, which activates mTOR, and is highly expressed in human cancers. We investigated the oncogenicity of human LAT1 introduced to NIH/3T3 cells by retrovirus infection. NIH/3T3 cell lines stably expressing human native (164C) or mutant (164S) LAT1 (naLAT1/3T3 or muLAT1/3T3, respectively) were established. We confirmed that endogenous mouse CD98hc forms a disulfide bond with exogenous human LAT1 in naLAT1/3T3, but not in muLAT1/3T3. Endogenous mouse CD98hc mRNA increased in both naNIH/3T3 and muLAT1/3T3, and a similar amount of exogenous human LAT1 protein was detected in both cell lines. Furthermore, naLAT1/3T3 and muLAT1/3T3 cell lines were evaluated for cell growth-related phenotypes (phosphorylation of ERK, cell-cycle progression) and cell malignancy-related phenotypes (anchorage-independent cell growth, tumor formation in nude mice). naLAT1/3T3 had stronger growth- and malignancy- related phenotypes than NIH/3T3 and muLAT1/3T3, suggesting the oncogenicity of native LAT1 through its interaction with CD98hc. Anti-LAT1 monoclonal antibodies significantly inhibited in vitro cell proliferation and in vivo tumor growth of naLAT1/3T3 cells in nude mice, demonstrating LAT1 to be a promising anti-cancer target.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity invitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes.

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation.

Short Communication: Impact of COVID-19 on Case Reporting for HTLV and HIV-2 in Spain.

The medical demand imposed by COVID-19 has distracted proper care of other illnesses. Herein, we report the impact on new diagnoses of HTLV-1, HTLV-2, and HIV-2 in Spain, where these infections are mostly driven by immigration flows from endemic regions. As expected, case reporting declined for all three retroviral infections with respect to prior years. Furthermore, late presentations were more common. The two major reasons for these observations were significant declines in the arrival of foreigners from endemic regions and a shift in medical resources to prioritize COVID-19.

Functions and Regulation of Endogenous Retrovirus Elements during Zygotic Genome Activation: Implications for Improving Somatic Cell Nuclear Transfer Efficiency.

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.

The use of molecular diagnostics of BLV in the study of various biological material

It is known that milk is one of the factors in the transmission of susceptible cattle to retroviral infection, in this case, induced bovine leukemia virus (BLV). On the other hand, one of the most important aspects of studying the biological properties of BLV is the potential ability of the virus to infect xenogenic species of animals, including humans, who consume animal products and are in close contact with cattle. Milk may contain leukocytes with integrated proviral DNA, which produce mature BLV virions and the mature virions themselves, which have come from the bloodstream. The use of molecular diagnostics of this biological material will make it possible to more effectively track contaminated dairy products on farms. However, the detection of BLV proviral DNA in milk is important not only for human health, but also for animals. Since some of the products are used directly on farms, for example for feeding young animals. In regard to this, we have undertaken optimization of the methodology for the study of milk for the presence of proviral DNA of BLV. The article presents the results of the analysis of milk from 5 seropositive cows of the Holstein breed by the method of molecular diagnostics real-time polymerase chain reaction (PCR-RT). When optimizing the method of sample preparation of milk, casein and other impurities that are inhibitors of the amplification reaction were removed, in order to reduce their influence, the samples were centrifuged under various conditions. Several nucleic acid isolation techniques were used to extract proviral DNA. Based on the results of the studies, a positive amplification result was recorded in 1 out of 5 animals. At the same time, this positive result was found in test tubes with complementary DNA (cDNA) samples, it means that RNA was isolated both from the "white ring" fraction with white blood cells and monocytes, and from the sediment cells.

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.

Effects of APOBEC3G's Cytidine Deaminase Activity on Retroviral Evolution

Apolipoprotein B editing complex (APOBEC3/A3) genes are found in mammalian cells. In primates, there are 7 APOBEC3 genes, namely, 3A, 3B, 3C, 3DE, 3F, 3G, and 3H. Previous research has shown that A3 proteins help to inhibit viral infection via their cytidine deaminase activity. However, it has also been found that A3 proteins could also lead to viral evolution, where viruses such as HIV (Human Immunodeficiency Virus) instead gain beneficial mutations that enable them to overcome the antiviral activity of A3 proteins, gain resistance to certain drugs used for treating viral infections and escape recognition by the immune system. This paper is a review article summarizing the role of A3G on viral infection and evolution, and the potential impact viral evolution could have in treatment of retroviral infections such as HIV.

Molecular Diagnosis of Koala Retrovirus (KoRV) in South Australian Koalas (Phascolarctos cinereus).

Simple SummaryKoala retrovirus (KoRV) is a significant threat to koalas across Australia. Koalas in northern koala populations (from New South Wales and Queensland) have KoRV inserted into their DNA and inherited to their offspring. Southern koala populations (from Victoria and South Australia) have KoRV infection spread through close contact of koalas. As such, there are koalas within South Australia that are not infected with KoRV. Accurate diagnosis of the infection of each koala is therefore fundamental for disease studies. Previous studies have shown differences in prevalence of different KoRV genes in the Mount Lofty Ranges Koala population; therefore, clarification is necessary. This study uses a large cohort (n = 216) and defines the diagnostic regions of the KoRV genome within the South Australian population. Using multiple molecular techniques, it demonstrates strong evidence for two clear groupings of koalas: KoRV positive and KoRV negative. Within this study, a population of 41% were shown to be KoRV positive and 57% were KoRV negative, with 2% inconclusive. This differentiation is of great importance when examining the clinical importance of KoRV infection within southern koalas.Koala retrovirus, a recent discovery in Australian koalas, is endogenised in 100% of northern koalas but has lower prevalence in southern populations, with lower proviral and viral loads, and an undetermined level of endogenisation. KoRV has been associated with lymphoid neoplasia, e.g., lymphoma. Recent studies have revealed high complexity in southern koala retroviral infections, with a need to clarify what constitutes positive and negative cases. This study aimed to define KoRV infection status in Mount Lofty Ranges koalas in South Australia using RNA-seq and proviral analysis (n = 216). The basis for positivity of KoRV was deemed the presence of central regions of the KoRV genome (gag 2, pol, env 1, and env 2) and based on this, 41% (89/216) koalas were positive, 57% (124/216) negative, and 2% inconclusive. These genes showed higher expression in lymph node tissue from KoRV positive koalas with lymphoma compared with other KoRV positive koalas, which showed lower, fragmented expression. Terminal regions (LTRs, partial gag, and partial env) were present in SA koalas regardless of KoRV status, with almost all (99.5%, 215/216) koalas positive for gag 1 by proviral PCR. Further investigation is needed to understand the differences in KoRV infection in southern koala populations.

Moloney Murine Leukemia Virus p12 Is Required for Histone Loading onto Retroviral DNAs.

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. IMPORTANCE Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus occurs only upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

Limited cross-species transmission and absence of mutations associated with SARS-CoV-2 adaptation in cats: A case study of infection in a small household setting.

In the present study, the course of SARS‐CoV‐2 natural infection in two asymptomatic cats, which were negative for immunosuppressive retroviral infections, is investigated. The source of the virus for the cats was their COVID‐19‐affected owner, with whom they were in continuous proximity in a small household setting. The owner's signs included fatigue, sneezing, anosmia and loss of taste, and diagnosis was confirmed 4 days after symptom onset. Oropharyngeal and faecal swabs were collected from the cats, to investigate the course of SARS‐CoV‐2 RNA concentrations, as well as the directionality of the chain of virus transmission. Both infected cats were real‐time RT‐PCR‐positive on various time‐points. Pharyngeal shedding of at least 6 days was observed in them, with high SARS‐CoV‐2 titres (> 7 Log10 copies/swab) on the first sampling time‐point, that is, 7 days after the onset of owner's clinical signs. In one cat, after the initial decline, slightly increasing virus titres were measured 3 to 6 days after the first real‐time RT‐PCR‐positive swab. Serological testing of this cat revealed absence of seroconversion. The course of viral RNA concentrations in the faecal swabs of the other cat was similar to that in its pharynx. The detected SARS‐CoV‐2 strains, from both infected cats and their owner, underwent whole‐genome sequencing, revealing the absence of emergence of cross‐species adaptive mutations in cats. The results support the notion that human SARS‐CoV‐2 strains are relatively well‐adapted to cats. It is still unclear whether asymptomatic animals could play a role in COVID‐19 epidemiology, in case of interaction with naïve animals and/or people. Our findings highlight difficulties in SARS‐CoV‐2 transmission to cats, as neither the two infected cats nor their owner was able to transmit the virus to a third cat living in the same small flat, despite their very close contact during the days corresponding to high virus shedding.

Prevalence of endoparasitic and viral infections in client-owned cats in metropolitan Bangkok, Thailand, and the risk factors associated with feline hookworm infections.

The prevalence of enteric parasites in cats in metropolitan Bangkok has not been updated in over 13years. The main objectives of this study include updating the prevalence of endoparasitism in client-owned cats, status of retroviral infections and determining the association between feline hookworm infection and possible risk factors. A total of 509 fecal samples were collected from client-owned cats in 2014-2015 and examined by a wet fecal mount technique. If additional sample remained, a PBS-ethyl acetate sedimentation was done (n=229), and ZnSO4 centrifugal flotation was also performed if there was sufficient remaining sample (n=105). At least one parasite was observed in 32.0% (163/509) of cats, with Ancylostoma being the most common intestinal parasite detected in 21.6% (110/509) of cats. Other parasitic infections detected by fecal examinations included Toxocara (6.9%; 35/509), Platynosomum (3.7%; 19/509), Cystoisospora (3.5%; 18/509), Taenia (2.9%; 15/509), Spirometra (1.6%; 8/509), Dipylidium (0.4%; 2/509), and Opisthorchis-like trematode (0.2%; 1/509). Examination for Giardia infection was conducted with the SNAP® Giardia Test, a coproantigen test, on a subset of the fecal samples (233/509) and revealed a positive result on 3.9% (9/233) of samples. Plasma samples were analyzed using the SNAP® Triple Test detecting antigens of Feline Leukemia Virus (FeLV) and Dirofilaria immitis while also detecting antibodies to Feline Immunodeficiency Virus (FIV). Antigens of FeLV and antibodies to FIV were found in 7.1% (19/269) and 5.2% (14/269) of cats, respectively. None of the cats were found to have circulating antigen of Dirofilaria immitis using this test. No association between retroviral and endoparasitic infections was found. From multivariable logistic regression examining associated factors, the ability of cats to access the outdoors (adjusted OR=3.22, 95% CI; 1.42-7.87) and having tapeworm segments or adult helminths in feces (adjusted OR=3.31, 95% CI; 1.34-8.21) were significantly associated with the finding of hookworm eggs in feces. This work presents the most up-to-date data on enteric feline parasite prevalence in the metropolitan Bangkok area from which fecal samples were directly collected from cats. Consequently, this study emphasizes that diagnosis of parasitic infections and the routine use of antiparasitic medications should be encouraged by veterinarians and to owners in order to reduce the reservoir of potentially zoonotic parasites.

Bone loss in hepatitis B virus-infected patients can be associated with greater osteoclastic activity independently of the retroviral use

Nucleoside/nucleotide analogs such as tenofovir, have been used as long-term therapy for the treatment of hepatitis B and side effects such as the reduction in bone mineral density have been associated with their use. To determine the relationships between bone, hormonal, biochemical, and mineral parameters in patients with hepatitis B treated with nucleoside/nucleotide antiviral. A cross-sectional study was conducted with 81 adult patients with chronic hepatitis B infection. Dual-energy X-ray absorptiometry (DXA) was performed to assess bone mineral density. Biochemical analyses were performed for osteocalcin, deoxypyridinoline, parathyroid hormone, vitamin D, IGF-1, TSH, testosterone, estradiol, FSH, transaminases, urea, creatinine, calcium, serum and urinary phosphorus, magnesium, and FGF-23, body composition was performed by DXA. Participants, both gender, were divided according to the use of antiretrovirals: Group1: 27 inactive virus carriers without medication; Group2: 27 patients using tenofovir; and Group3: 27 patients using lamivudine or entecavir. DXA readings diagnosed osteopenia in the lumbar spine for 7.4% of individuals in Group1, 15% in Group2, and 3.7% in Group3. For all groups, we observed normal values in bone formation markers, osteocalcin levels as well as parathyroid hormone, insulin growth factor 1, and FGF-23. In all groups, we found increased levels of urinary deoxypyridinoline, a bone resorption marker. Increased levels in the bone resorption markers indicated a high resorptive activity of bone tissue. These data suggested high resorption activity of bone tissue in hepatitis B virus-infected patients independent of the use of antiretrovirals.

Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses.

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Human Endogenous Retroviruses (HERVs) and Mammalian Apparent LTRs Retrotransposons (MaLRs) Are Dynamically Modulated in Different Stages of Immunity.

Simple SummaryHuman Endogenous retroviruses (HERVs) and Mammalian Apparent LTRs Retrotransposons (MaLRs) are remnants of ancient retroviral infections that make up about 8% of the human genome. HERV and MaLR expression is regulated in immunity, and, in particular, is known for their up-regulation after innate immune activation. In this work, we analyzed the differential expression of HERVs and MaLRs in different stages of immunity, triggered by the administration of an inactivated vaccine.Human Endogenous retroviruses (HERVs) and Mammalian Apparent LTRs Retrotransposons (MaLRs) are remnants of ancient retroviral infections that represent a large fraction of our genome. The HERV and MaLR transcriptional activity is regulated in developmental stages, adult tissues, and pathological conditions. In this work, we used a bioinformatics approach based on RNA-sequencing (RNA-seq) to study the expression and modulation of HERVs and MaLR in a scenario of activation of the immune response. We analyzed transcriptome data from subjects before and after the administration of an inactivated vaccine against the Hantaan orthohantavirus, the causative agent of Korean hemorrhagic fever, to investigate the HERV and MaLR expression and differential expression in response to the administration of the vaccine. Specifically, we described the HERV transcriptome in PBMCs and identified HERV and MaLR loci differentially expressed after the 2nd, 3rd, and 4th inactivated vaccine administrations. We found that the expression of 545 HERV and MaLR elements increased in response to the vaccine and that the activation of several individual HERV and MaLR loci is specific for each vaccine administration and correlated to different genes and immune-related pathways.

Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy.

Koala populations are currently declining and under threat from koala retrovirus (KoRV) infection both in the wild and in captivity. KoRV is assumed to cause immunosuppression and neoplastic diseases, favoring chlamydiosis in koalas. Currently, 10 KoRV subtypes have been identified, including an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). The host’s immune response acts as a safeguard against pathogens. Therefore, a proper understanding of the immune response mechanisms against infection is of great importance for the host’s survival, as well as for the development of therapeutic and prophylactic interventions. A vaccine is an important protective as well as being a therapeutic tool against infectious disease, and several studies have shown promise for the development of an effective vaccine against KoRV. Moreover, CRISPR/Cas9-based genome editing has opened a new window for gene therapy, and it appears to be a potential therapeutic tool in many viral infections, which could also be investigated for the treatment of KoRV infection. Here, we discuss the recent advances made in the understanding of the immune response in KoRV infection, as well as the progress towards vaccine development against KoRV infection in koalas.

Immunization by exposure to live virus (SIVmne/HIV-2287) during antiretroviral drug prophylaxis may reduce risk of subsequent viral challenge.

Rationale/Study designA major challenge in the development of HIV vaccines is finding immunogens that elicit protection against a broad range of viral strains. Immunity to a narrow range of viral strains may protect infants of HIV-infected women or partners discordant for HIV. We hypothesized that immunization to the relevant viral variants could be achieved by exposure to infectious virus during prophylaxis with antiretroviral drugs. To explore this approach in an animal model, macaques were exposed to live virus (SIVmne or HIV-2287) during prophylaxis with parenteral tenofovir and humoral and cellular immune responses were quantified. Subsequently, experimental animals were challenged with homologous virus to evaluate protection from infection, and if infection occurred, the course of disease was compared to control animals. Experimental animals uninfected with SIVmne were challenged with heterologous HIV-2287 to assess resistance to retroviral infection.Methodology/Principal findingsJuvenile female Macaca nemestrina (N = 8) were given ten weekly intravaginal exposures with either moderately (SIVmne) or highly (HIV-2287) pathogenic virus during tenofovir prophylaxis. Tenofovir protected all 8 experimental animals from infection, while all untreated control animals became infected. Specific non-neutralizing antibodies were elicited in blood and vaginal secretions of experimental animals, but no ELISPOT responses were detected. Six weeks following the cessation of tenofovir, intravaginal challenge with homologous virus infected 2/4 (50%) of the SIVmne-immunized animals and 4/4 (100%) of the HIV-2287-immunized animals. The two SIVmne-infected and 3 (75%) HIV-2287-infected had attenuated disease, suggesting partial protection.Conclusions/SignificanceRepeated exposure to SIVmne or HIV-2287, during antiretroviral prophylaxis that blocked infection, induced binding antibodies in the blood and mucosa, but not neutralizing antibodies or specific cellular immune responses. Studies to determine whether antibodies are similarly induced in breastfeeding infants and sexual partners discordant for HIV infection and receiving pre-exposure antiretroviral prophylaxis are warranted, including whether these antibodies appear to confer partial or complete protection from infection.

Oncolytic Foamy Virus: Generation and Properties of a Nonpathogenic Replicating Retroviral Vector System That Targets Chronically Proliferating Cancer Cells

Nonpathogenic retroviruses of the Spumaretrovirinae subfamily can persist long-term in the cytoplasm of infected cells, completing their lifecycle only after the nuclear membrane dissolves at the time of cell division. Since the targeting of slowly dividing cancer cells remains an unmet need in oncolytic virotherapy we constructed a replication competent Foamy Virus vector (oFV) from the genomes of two chimpanzee Simian Foamy Viruses (PAN1 and PAN2) and inserted a GFP transgene in place of the bel-2 open reading frame. oFV-GFP infected and propagated with slow kinetics in multiple human tumor cell lines, inducing a syncytial cytopathic effect. Infection of growth arrested MRC5 cells was not productive, but oFV genomes persisted in the cytoplasm and the productive viral lifecycle resumed when cell division was later restored. In vivo, the virus propagated extensively in intraperitoneal ovarian cancer xenografts, slowing tumor growth, significantly prolonging survival of the treated mice and sustaining GFP transgene expression for at least 45 days. Our data indicate that oFV is a promising new replication-competent viral and gene delivery platform for efficient targeting of the most fundamental trait of cancer cells, their ability to sustain chronic proliferation.Significance:The infectivity of certain retroviruses is limited to dividing cells, which makes them attractive tools for targeting cancer cell proliferation. Previously developed replication-competent gammaretroviral vectors spread efficiently in rapidly dividing cancer cells, but not in cancer cells that divide more slowly. In contrast to rapidly proliferating transplantable mouse tumors, slow proliferation is a hallmark of human cancers and may have contributed to the clinical failure of the preclinically promising Murine Leukemia Virus vector Toca511 which failed to show efficacy in a phase 3 clinical trial in patients with glioblastoma. The studies presented in our manuscript show that oncolytic Foamy Virus (oFV) vectors are capable of persisting unintegrated in quiescent cells and resuming their life cycle once the cells start dividing again. This property of oFVs, together with their lack of pathogenicity and their ability to catalyze the fusion of infected cancer cells, makes them an attractive platform for further investigation.

Prevalence of pathological lesions diagnosed by cytology in cats, with association of diagnosis to age, breed and gender

Cytology is the diagnostic procedure of the microscopic evaluation of cells. It is becoming increasingly important and more frequently used in veterinary diagnostics, having many advantages including simplicity, speed and low cost. To determine the pathological changes diagnosed by cytology in cats, as well as the distribution of age, breed and gender in the diagnosed changes, we performed a retrospective study on slides submitted to the Department of Veterinary Pathology for routine cytological examination. The archive was searched for all feline cytology slides submitted from 2009 to 2018. All the slides were re-evaluated and classified into one of the four pathological processes: ‘neoplasia’, ‘inflammation’, ‘other condition’ or ‘non-diagnostic sample’. Breed, age, gender and the tissue from which the lesion was sampled were noted from the submission form, and statistically analyzed. The most frequent type of pathological process diagnosed was neoplasia, which reflects the high prevalence of neoplastic diseases in cats reported in literature data. Pathological changes were mostly diagnosed in domestic shorthaired cats of both sexes, with an average age of 8.4 years, but no breed, age or gender predisposition was found. The most evaluated tissue was skin, probably due to its accessibility and the ease of obtaining a sample from skin lesions. The most frequent neoplasia were malignant and the most frequent diagnosis was round cell neoplasia. Cats affected with round cell neoplasia had a significantly lower average age (7.3 years) than cats diagnosed with epithelial and mesenchymal neoplasia (9.9 and 10.3 years, respectively), probably reflecting the common retroviral infection in Croatian cats.