Evolution of Endogenous Retroviruses in the Subfamily of Caprinae.

The interest in endogenous retroviruses (ERVs) has been fueled by their impact on the evolution of the host genome. In this study, we used multiple pipelines to conduct a de novo exploration and annotation of ERVs in 13 species of the Caprinae subfamily. Through analyses of sequence identity, structural organization, and phylogeny, we defined 28 ERV groups within Caprinae, including 19 gamma retrovirus groups and 9 beta retrovirus groups. Notably, we identified four recent and potentially active groups prevalent in the Caprinae genomes. Additionally, our investigation revealed that most long noncoding genes (lncRNA) and protein-coding genes (PC) contain ERV-derived sequences. Specifically, we observed that ERV-derived sequences were present in approximately 75% of protein-coding genes and 81% of lncRNA genes in sheep. Similarly, in goats, ERV-derived sequences were found in approximately 74% of protein-coding genes and 75% of lncRNA genes. Our findings lead to the conclusion that the majority of ERVs in the Caprinae genomes can be categorized as fossils, representing remnants of past retroviral infections that have become permanently integrated into the genomes. Nevertheless, the identification of the Cap_ERV_20, Cap_ERV_21, Cap_ERV_24, and Cap_ERV_25 groups indicates the presence of relatively recent and potentially active ERVs in these genomes. These particular groups may contribute to the ongoing evolution of the Caprinae genome. The identification of putatively active ERVs in the Caprinae genomes raises the possibility of harnessing them for future genetic marker development.

FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

Contrasting segregation patterns among endogenous retroviruses across the koala population.

Koalas (Phascolarctos cinereus) have experienced a history of retroviral epidemics leaving their trace as heritable endogenous retroviruses (ERVs) in their genomes. A recently identified ERV lineage, named phaCin-ß, shows a pattern of recent, possibly current, activity with high insertional polymorphism in the population. Here, we investigate geographic patterns of three focal ERV lineages of increasing estimated ages, from the koala retrovirus (KoRV) to phaCin-ß and to phaCin-ß-like, using the whole-genome sequencing of 430 koalas from the Koala Genome Survey. Thousands of ERV loci were found across the population, with contrasting patterns of polymorphism. Northern individuals had thousands of KoRV integrations and hundreds of phaCin-ß ERVs. In contrast, southern individuals had higher phaCin-ß frequencies, possibly reflecting more recent activity and a founder effect. Overall, our findings suggest high ERV burden in koalas, reflecting historic retrovirus-host interactions. Importantly, the ERV catalogue supplies improved markers for conservation genetics in this endangered species.

GWAS reveals determinants of mobilization rate and dynamics of an active endogenous retrovirus of cattle.

Five to ten percent of mammalian genomes is occupied by multiple clades of endogenous retroviruses (ERVs), that may count thousands of members. New ERV clades arise by retroviral infection of the germline followed by expansion by reinfection and/or retrotransposition. ERV mobilization is a source of deleterious variation, driving the emergence of ERV silencing mechanisms, leaving "DNA fossils". Here we show that the ERVK[2-1-LTR] clade is still active in the bovine and a source of disease-causing alleles. We develop a method to measure the rate of ERVK[2-1-LTR] mobilization, finding an average of 1 per ~150 sperm cells, with >10-fold difference between animals. We perform a genome-wide association study and identify eight loci affecting ERVK[2-1-LTR] mobilization. We provide evidence that polymorphic ERVK[2-1-LTR] elements in four of these loci cause the association. We generate a catalogue of full length ERVK[2-1-LTR] elements, and show that it comprises 15% of C-type autonomous elements, and 85% of D-type non-autonomous elements lacking functional genes. We show that >25% of the variance of mobilization rate is determined by the number of C-type elements, yet that de novo insertions are dominated by D-type elements. We propose that D-type elements act as parasite-of-parasite gene drives that may contribute to the observed demise of ERV elements.

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection.

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future.

Investigation of koala retrovirus in captive koalas with pneumonia and comparative analysis of subtype distribution.

This study focused on the involvement of koala retrovirus (KoRV) in pneumonia in koalas. Three deceased pneumonic koalas from a Japanese zoo were examined in this study. Hematological and histopathological findings were assessed, and KoRV proviral DNA loads in the blood and tissues were compared with those of eight other KoRV-infected koalas from different zoos. Demographic data and routine blood profiles were collected, and blood and tissue samples were analyzed to rule out concurrent infections in pneumonic koalas. KoRV subtyping and measurement of the KoRV proviral DNA load were performed by polymerase chain reaction (PCR) using specific primers targeting the pol and env genes. The results showed that the koalas had histopathologically suppurative and fibrinous pneumonia. Chlamydiosis was not detected in any of the animals. PCR analysis revealed KoRV-A, -B, and -C infections in all koalas, except for animals K10-11, which lacked KoRV-B. Significant variations in the proviral DNA loads of these KoRV subtypes were observed in all tissues and disease groups. Most tissues showed reduced KoRV loads in koalas with pneumonia, except in the spleen, which had significantly higher loads of total KoRV (2.54 × 107/µg DNA) and KoRV-A (4.74 × 107/µg DNA), suggesting potential immunosuppression. This study revealed the intricate dynamics of KoRV in various tissues, indicating its potential role in koala pneumonia via immunosuppression and opportunistic infections. Analysis of the levels of KoRV proviral DNA in different tissues will shed light on viral replication and the resulting pathogenesis in future studies.

CROI 2023: Summary of Basic Science Research in HIV.

The 2023 Conference on Retroviruses and Opportunistic Infections (CROI) represented the first fully in-person conference since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic began. CROI continues as the premier conference in which delegates can appraise themselves of almost every facet of HIV/AIDS research as well as emerging and re-emerging pathogens such as SARS-CoV-2 and mpox. The return to an in-person format is particularly important for early-stage investigators, who were faced with challenges of advancing their independent research careers during the SARS-CoV-2 pandemic. The personnel interactions and face-to-face meetings between junior investigators and their peers enable collaboration that is important in the academic development process. A very packed program showcased research advances in basic research, clinical, and epidemiology/public health endeavors around HIV and other pandemic viruses. Session presentation summaries, themed discussion sessions, and scientific workshops condense and assimilate specific areas of research that are particularly useful for delegates who want to see the state of research in areas that may be outside their specific areas of interest. The conference organizers drew on more than 1000 accepted abstracts to assemble a dynamic and engaging program that was appealing to infectious disease researchers worldwide.

CROI 2023: Metabolic and Other Complications of HIV Infection.

Comorbid conditions have major impacts on the health, quality of life, and survival of people with HIV, particularly as they age. The 2023 Conference on Retroviruses and Opportunistic Infections (CROI) featured excellent science related to specific comorbidities, such as cardiovascular disease (CVD), cancer, and obesity. Studies investigating factors that may contribute to CVD, such as mental health disorders, antiretroviral therapies, and activation of hormonal pathways, were featured prominently. Other studies sought to understand the epidemiology of non-AIDS-defining cancers in people with HIV. As at previous CROI conferences, weight gain attributable to antiretroviral therapies was a major theme, and several abstracts focused on the important question of whether weight decreases after discontinuation of antiretroviral therapy (ART) regimens associated with weight gain. This review focuses on abstracts presented at CROI 2023 in these areas, highlighting those with the most clinical impact.

CROI 2023: Neuropsychiatric Complications in People With HIV.

The 2023 Conference on Retroviruses and Opportunistic Infections (CROI) featured new and impactful findings about neuropsychiatric complications in people with HIV and other infections. Reports included new evidence of (a) the importance of myeloid cells in the pathogenesis of HIV disease in the central nervous system, including as an HIV reservoir; (b) eukaryotic and prokaryotic viruses in cerebrospinal fluid during suppressive antiretroviral therapy; (c) the influence of sex on pathogenesis, including in novel neuropsychiatric biotypes identified by machine learning and other methods;(d) premature aging in people with HIV, including the brain-age gap observed on magnetic resonance imaging; (e) cellular and soluble biomarkers of neuropsychiatric complications in people with HIV; and (f) the neurotoxicity of certain antiretroviral drugs. This review summarizes these and other new findings and highlights new research directions for the neuro-HIV field.

A single dominant locus restricts retrovirus replication in YBR/Ei mice.

Differential responses to viral infections are influenced by the genetic makeup of the host. Studies of resistance to retroviruses in human populations are complicated due to the inability to conduct proof-of-principle studies. Inbred mouse lines, which have a range of susceptible phenotypes to retroviruses, are an ideal tool to identify and characterize mechanisms of resistance and define their genetic underpinnings. YBR/Ei mice become infected with Mouse Mammary Tumor Virus, a mucosally transmitted murine retrovirus, but eliminate the virus from their pedigrees. Virus elimination correlates with a lack of virus-specific neonatal oral tolerance, which is a major mechanism for blocking the anti-virus response in susceptible mice. Virus control is unrelated to virus-neutralizing antibodies, cytotoxic CD8+ T cells, NK cells, and NK T cells, which are the best characterized mechanisms of resistance to retroviruses. We identified a single, dominant locus that controls the resistance mechanism, which we provisionally named attenuation of virus titers (Avt) and mapped to the distal region of chromosome 18. IMPORTANCE Elucidation of the mechanism that mediates resistance to retroviruses is of fundamental importance to human health, as it will ultimately lead to knowledge of the genetic differences among individuals in susceptibility to microbial infections.

Characterizing the Pathogenicity and Immunogenicity of Simian Retrovirus Subtype 8 (SRV-8) Using SRV-8-Infected Cynomolgus Monkeys.

Simian retrovirus subtype 8 (SRV-8) infections have been reported in cynomolgus monkeys (Macaca fascicularis) in China and America, but its pathogenicity and immunogenicity are rarely reported. In this work, the SRV-8-infected monkeys were identified from the monkeys with anemia, weight loss, and diarrhea. To clarify the impact of SRV-8 infection on cynomolgus monkeys, infected monkeys were divided into five groups according to disease progression. Hematoxylin (HE) staining and viral loads analysis showed that SRV-8 mainly persisted in the intestine and spleen, causing tissue damage. Additionally, the dynamic variations of blood routine indexes, innate and adaptive immunity, and the transcriptomic changes in peripheral blood cells were analyzed during SRV-8 infection. Compared to uninfected animals, red blood cells, hemoglobin, and white blood cells were reduced in SRV-8-infected monkeys. The percentage of immune cell populations was changed after SRV-8 infection. Furthermore, the number of hematopoietic stem cells decreased significantly during the early stages of SRV-8 infection, and returned to normal levels after antibody-mediated viral clearance. Finally, global transcriptomic analysis in PBMCs from SRV-8-infected monkeys revealed distinct gene expression profiles across different disease stages. In summary, SRV-8 infection can cause severe pathogenicity and immune disturbance in cynomolgus monkeys, and it might be responsible for fatal virus-associated immunosuppressive syndrome.

Regulatory T cells suppress the motility of cytotoxic T cells in Friend retrovirus-infected mice.

Antiviral immunity often requires CD8+ cytotoxic T lymphocytes (CTLs) that actively migrate and search for virus-infected targets. Regulatory T cells (Tregs) have been shown to suppress CTL responses, but it is not known whether this is also mediated by effects on CTL motility. Here, we used intravital 2-photon microscopy in the Friend retrovirus (FV) mouse model to define the impact of Tregs on CTL motility throughout the course of acute infection. Virus-specific CTLs were very motile and had frequent short contacts with target cells at their peak cytotoxic activity. However, when Tregs were activated and expanded in late-acute FV infection, CTLs became significantly less motile and contacts with target cells were prolonged. This phenotype was associated with development of functional CTL exhaustion. Tregs had direct contacts with CTLs in vivo and, importantly, their experimental depletion restored CTL motility. Our findings identify an effect of Tregs on CTL motility as part of their mechanism of functional impairment in chronic viral infections. Future studies must address the underlying molecular mechanisms.

CROI 2023: Advances in Antiviral Therapy in HIV and Viral Hepatitis.

Several innovative methods were presented at the 2023 Conference on Retroviruses and Opportunistic Infections (CROI) targeting different aspects of the HIV care continuum to improve testing, linkage to care, and viral suppression. Some of these approaches were directed at more vulnerable groups, such as pregnant women, adolescents, and individuals who inject drugs. In contrast was the devastating impact of the COVID-19 pandemic, with negative outcomes on HIV viral load suppression and retention in care. Data were presented on hepatitis B virus (HBV) suppression showing that tenofovir alafenamide (TAF)/emtricitabine (FTC)/bictegravir (BIC) may be superior to tenofovir disoproxil fumarate/FTC plus dolutegravir in suppressing HBV in HIV/HBV-coinfected individuals. A pilot study examining a 4-week trial of direct-acting antiviral therapy to treat hepatitis C in recently infected individuals showed lower rates of sustained virologic response at 12 weeks than longer courses. Additional data were presented on the use of long-acting cabotegravir/rilpivirine, comparing this regimen with oral TAF/FTC/BIC and the use of long-acting cabotegravir/rilpivirine in those with viremia. Data were presented on a novel strategy of lenacapavir with 2 broadly neutralizing antibodies given every 6 months as maintenance antiretroviral therapy (ART). Data were presented on improving HIV care outcomes in adolescents, interventions to prevent mother-to-child transmission, and HIV reservoirs in children and adolescents. Data were also presented on interactions between ART and hormonal contraception, as well as ART-related weight gain and impact on pregnancy. A study examining BIC pharmacokinetics in pregnancy was presented, as well as retrospective data on outcomes of adolescents receiving TAF/FTC/BIC.

CROI 2023: Epidemiologic Trends and Prevention for HIV and Other Sexually Transmitted Infections.

At the 2023 Conference on Retroviruses and Opportunistic Infections (CROI), several investigators used tests of recent HIV infection to track which populations are currently most heavily impacted by HIV and to estimate HIV infection rates in those populations. Assisted partner notification for HIV was successfully applied for spouses of persons with HIV and sexual and injection partners of people who inject drugs; however, delays in linkage to care were seen for non-spousal partners in one study. Lack of awareness of HIV positive status remains an issue in various populations; several presentations focused on novel strategies for improving HIV testing uptake in these populations. Doxycycline administered as 200 mg post sexual exposure significantly reduced the risk of syphilis, chlamydia, and gonorrhea infection in men who have sex with men but did not prevent bacterial sexually transmitted infections (STIs) in cis-gender women; reasons for this discrepancy are currently being explored. Although oral HIV preexposure prophylaxis (PrEP) is increasingly being used in populations in greatest need of prevention tools, PrEP uptake and persistence remain low in a number of key populations, including people who inject drugs. Several innovative delivery models show early promise in addressing gaps along the PrEP continuum. The successful use of injectable cabotegravir PrEP in several populations was presented at this conference, although uptake remains low globally. The pipeline of novel long-acting and rapid-onset PrEP agents appears to be robust, including implants, vaginal rings, and topical inserts, with several presentations focusing on preclinical and early clinical trials.

CROI 2023: Acute and Post-Acute COVID-19.

Studies of acute and post-acute COVID-19 were presented at the 2023 Conference on Retroviruses and Opportunistic Infections (CROI). Early treatment with ensitrelvir, a novel protease inhibitor, hastened viral clearance and symptom resolution during coronavirus disease 2019 (COVID-19) and appeared to reduce the prevalence of long COVID symptoms. The development of novel agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including those with broader sarbecovirus activity such as anti-angiotensin-converting enzyme 2 monoclonal antibodies, is underway. A growing understanding of the pathophysiology of long COVID has provided several potential therapeutic targets for individuals experiencing this condition. Efforts to understand COVID-19 in people with HIV have led to novel insights into the biology and natural history of SARS-CoV-2 coinfection in this vulnerable subpopulation. These and other studies are summarized herein.

CROI 2023: Epidemiology, Diagnosis, and Management of MPox.

The 2023 Conference on Retroviruses and Opportunistic Infections (CROI) emphasized emerging infectious diseases such as COVID-19 and mpox. Despite emerging from countries in which it was endemic only 9 months before the conference, mpox was well covered, with more than 60 presentations addressing various topics. There was a focus on the rapid development and implementation of tests to reduce the time to diagnosis, as well as multiplex panels to increase the accuracy of differential diagnosis. Presenters also highlighted the ability to diagnose mpox from multiple compartments, such as with rectal and pharyngeal swabs, and provided crucial information on the duration of positivity that may impact isolation requirements. Clinical experiences were described, including risk factors for severe disease and syndemic management. High rates of concomitant sexually transmitted infection (STI) were reported. Finally, prevention was a key topic, with presenters pointing to the contributions of individual behavioral changes and vaccine efficacy to reducing new cases.

Jawless vertebrates do not escape retrovirus infection.

On occasion, retroviruses infect the genome of germline cell, forming endogenous retroviruses (ERVs), which provide molecular fossils for studying the deep evolution of retroviruses. While ERVs have been extensively characterized in the genomes of jawed vertebrates, much remains contentious and unexplored about the diversity and evolution of ERVs within jawless vertebrates. Here, we report the discovery of a novel ERV lineage, designated EbuERVs, in the genome of a hagfish Eptatretus burgeri. Phylogenetic analyses show that EbuERVs pertain to epsilon-retroviruses and might have derived from cross-species transmission from jawed vertebrates. EbuERVs are estimated to have invaded in the hagfish genome at least tens of millions of years ago. Evolutionary dynamics analyses indicate that EbuERVs might have experienced one proliferation peak and have been not active in transposition anymore. However, some EbuERVs can transcribe in embryo and might serve as lncRNA. Overall, these findings expand the distribution of retroviruses from jawed vertebrates to jawless vertebrates.

The Involvement of Ubiquitination and SUMOylation in Retroviruses Infection and Latency.

Retroviruses, especially the pathogenic human immunodeficiency virus type 1 (HIV-1), have severely threatened human health for decades. Retroviruses can form stable latent reservoirs via retroviral DNA integration into the host genome, and then be temporarily transcriptional silencing in infected cells, which makes retroviral infection incurable. Although many cellular restriction factors interfere with various steps of the life cycle of retroviruses and the formation of viral latency, viruses can utilize viral proteins or hijack cellular factors to evade intracellular immunity. Many post-translational modifications play key roles in the cross-talking between the cellular and viral proteins, which has greatly determined the fate of retroviral infection. Here, we reviewed recent advances in the regulation of ubiquitination and SUMOylation in the infection and latency of retroviruses, focusing on both host defense- and virus counterattack-related ubiquitination and SUMOylation system. We also summarized the development of ubiquitination- and SUMOylation-targeted anti-retroviral drugs and discussed their therapeutic potential. Manipulating ubiquitination or SUMOylation pathways by targeted drugs could be a promising strategy to achieve a "sterilizing cure" or "functional cure" of retroviral infection.

Genetic Differences between 129S Substrains Affect Antiretroviral Immune Responses.

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype in 129S1 mice is controlled by two dominant loci that are independent from known MLV resistance genes. We also show that production of anti-MLV antibodies in 129S7 mice, but not 129S1 mice, is independent of interferon gamma signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses. IMPORTANCE Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.

Feline Foamy Virus Transmission in Tsushima Leopard Cats (Prionailurus bengalensis euptilurus) on Tsushima Island, Japan.

Tsushima leopard cats (TLC; Prionailurus bengalensis euptilurus) only inhabit Tsushima Island, Nagasaki, Japan and are critically endangered and threatened by infectious diseases. The feline foamy virus (FFV) is widely endemic in domestic cats. Therefore, its transmission from domestic cats to TLCs may threaten the TLC population. Thus, this study aimed to assess the possibility that domestic cats could transmit FFV to TLCs. Eighty-nine TLC samples were screened, and FFV was identified in seven (7.86%). To assess the FFV infection status of domestic cats, 199 domestic cats were screened; 14.07% were infected. The phylogenetic analysis revealed that the FFV partial sequence from domestic cats and TLC sequences clustered in one clade, suggesting that the two populations share the same strain. The statistical data minimally supported the association between increased infection rate and sex (p = 0.28), indicating that FFV transmission is not sex dependent. In domestic cats, a significant difference was observed in FFV detection in feline immunodeficiency virus (p = 0.002) and gammaherpesvirus1 infection statuses (p = 0.0001) but not in feline leukemia virus infection status (p = 0.21). Monitoring FFV infection in domestic cats and TLC populations is highly recommended as part of TLC surveillance and management strategies.