The gonadal niche safeguards human fetal germline cell development following maternal SARS-CoV-2 infection.

During pregnancy, germline development is vital for maintaining the continuation of species. Recent studies have shown increased pregnancy risks in COVID-19 patients at the perinatal stage. However, the potential consequence of infection for reproductive quality in developing fetuses remains unclear. Here, we analyze the transcriptome and DNA methylome of the fetal germline following maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We find that infection at early gestational age, a critical period of human primordial germ cell specification and epigenetic reprogramming, trivially affects fetal germ cell (FGC) development. Additionally, FGC-niche communications are not compromised by maternal infection. Strikingly, both general and SARS-CoV-2-specific immune pathways are greatly activated in gonadal niche cells to protect FGCs from maternal infection. Notably, there occurs an "in advance" development tendency in FGCs after maternal infection. Our study provides insights into the impacts of maternal SARS-CoV-2 infection on fetal germline development and serves as potential clinical guidance for future pandemics.

Human reproduction is regulated by retrotransposons derived from ancient Hominidae-specific viral infections.

Germ cells are essential to pass DNA from one generation to the next. In human reproduction, germ cell development begins with the specification of primordial germ cells (PGCs) and a failure to specify PGCs leads to human infertility. Recent studies have revealed that the transcription factor network required for PGC specification has diverged in mammals, and this has a significant impact on our understanding of human reproduction. Here, we reveal that the Hominidae-specific Transposable Elements (TEs) LTR5Hs, may serve as TEENhancers (TE Embedded eNhancers) to facilitate PGC specification. LTR5Hs TEENhancers become transcriptionally active during PGC specification both in vivo and in vitro with epigenetic reprogramming leading to increased chromatin accessibility, localized DNA demethylation, enrichment of H3K27ac, and occupation of key hPGC transcription factors. Inactivation of LTR5Hs TEENhancers with KRAB mediated CRISPRi has a significant impact on germ cell specification. In summary, our data reveals the essential role of Hominidae-specific LTR5Hs TEENhancers in human germ cell development.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Recommendations for reducing the risk of viral transmission during fertility treatment with the use of autologous gametes: a committee opinion.

Sexually transmitted infections are of major concern to reproductive specialists. Heading the list are human immunodeficiency virus types 1 and 2 and hepatitis B and C viruses. These pathogens, which may cause incurable chronic infections, can be transmitted through assisted reproductive technologies and from infected mothers to the fetus or newborn. This document replaces the document of the same name last published in 2013 (Fertil Steril 2013;99:340-6).

Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV.

Viruses have colonized the germ line of our ancestors on several occasions during evolution, leading to the integration in the human genome of viral sequences from over 30 retroviral groups and a few nonretroviruses. Among the recently emerged viruses infecting humans, several target the testis (e.g., human immunodeficiency virus [HIV], Zika virus, and Ebola virus). Here, we aimed to investigate whether human testicular germ cells (TGCs) can support integration by HIV, a contemporary retrovirus that started to spread in the human population during the last century. We report that albeit alternative receptors enabled HIV-1 binding to TGCs, HIV virions failed to infect TGCs in vitro Nevertheless, exposure of TGCs to infected lymphocytes, naturally present in the testis from HIV+ men, led to HIV-1 entry, integration, and early protein expression. Similarly, cell-associated infection or bypassing viral entry led to HIV-1 integration in a spermatogonial cell line. Using DNAscope, HIV-1 and simian immunodeficiency virus (SIV) DNA were detected within a few TGCs in the testis from one infected patient, one rhesus macaque, and one African green monkey in vivo Molecular landscape analysis revealed that early TGCs were enriched in HIV early cofactors up to integration and had overall low antiviral defenses compared with testicular macrophages and Sertoli cells. In conclusion, our study reveals that TGCs can support the entry and integration of HIV upon cell-associated infection. This could represent a way for this contemporary virus to integrate into our germ line and become endogenous in the future, as happened during human evolution for a number of viruses.IMPORTANCE Viruses have colonized the host germ line on many occasions during evolution to eventually become endogenous. Here, we aimed at investigating whether human testicular germ cells (TGCs) can support such viral invasion by studying HIV interactions with TGCs in vitro Our results indicate that isolated primary TGCs express alternative HIV-1 receptors, allowing virion binding but not entry. However, HIV-1 entered and integrated into TGCs upon cell-associated infection and produced low levels of viral proteins. In vivo, HIV-1 and SIV DNA was detected in a few TGCs. Molecular landscape analysis showed that TGCs have overall weak antiviral defenses. Altogether, our results indicate that human TGCs can support HIV-1 early replication, including integration, suggesting potential for endogenization in future generations.

Viruses in the reproductive tract: On their way to the germ line?

Studies of vertebrate genomes have indicated that all species contain in their chromosomes stretches of DNA with sequence similarity to viral genomes. How such 'endogenous' viral elements (EVEs) ended up in host genomes is usually explained in general terms such as 'they entered the germ line at some point during evolution'. This seems a correct statement, but is also rather imprecise. The vast number of endogenous viral sequences suggest that common routes to the 'germ line' may exist, as relying on chance alone may not easily explain the abundance of EVEs in modern mammalian genomes. An increasing number of virus types have been detected in human semen and a growing number of studies have reported on viral infections that cause male infertility or subfertility and on viral infections that threaten in vitro fertilisation practices. Thus, it is timely to survey the pathway(s) that viruses can use to gain access to the human germ line. Embryo transfer and semen quality studies in livestock form another source of relevant information because virus infection during reproduction is clearly unwanted, as is the case for the human situation. In this review, studies on viruses in the male and female reproductive tract and in the early embryo will be discussed to propose a plausible viral route to the mammalian germ line.

The Cellular Impact of the ZIKA Virus on Male Reproductive Tract Immunology and Physiology.

Zika virus (ZIKV) has been reported by several groups as an important virus causing pathological damage in the male reproductive tract. ZIKV can infect and persist in testicular somatic and germ cells, as well as spermatozoa, leading to cell death and testicular atrophy. ZIKV has also been detected in semen samples from ZIKV-infected patients. This has huge implications for human reproduction. Global scientific efforts are being applied to understand the mechanisms related to arboviruses persistency, pathogenesis, and host cellular response to suggest a potential target to develop robust antiviral therapeutics and vaccines. Here, we discuss the cellular modulation of the immunologic and physiologic properties of the male reproductive tract environment caused by arboviruses infection, focusing on ZIKV. We also present an overview of the current vaccine effects and therapeutic targets against ZIKV infection that may impact the testis and male fertility.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Molecular Properties and Evolutionary Origins of a Parvovirus-Derived Myosin Fusion Gene in Guinea Pigs.

Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein.IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.

Position statement on West Nile virus: a committee opinion.

Although there is currently no definitive evidence linking West Nile virus (WNV) transmission with reproductive cells, it is recommended that practitioners defer gamete donors who have confirmed or suspected WNV infections. This document replaces the previously published document of the same name, last published in 2016 (Fertil Steril 2016;105:e9-10).

Zika virus infects human testicular tissue and germ cells.

Zika virus (ZIKV) is a teratogenic mosquito-borne flavivirus that can be sexually transmitted from man to woman. The finding of high viral loads and prolonged viral shedding in semen suggests that ZIKV replicates within the human male genital tract, but its target organs are unknown. Using ex vivo infection of organotypic cultures, we demonstrated here that ZIKV replicates in human testicular tissue and infects a broad range of cell types, including germ cells, which we also identified as infected in semen from ZIKV-infected donors. ZIKV had no major deleterious effect on the morphology and hormonal production of the human testis explants. Infection induced a broad antiviral response but no IFN upregulation and minimal proinflammatory response in testis explants, with no cytopathic effect. Finally, we studied ZIKV infection in mouse testis and compared it to human infection. This study provides key insights into how ZIKV may persist in semen and alter semen parameters, as well as a valuable tool for testing antiviral agents.

Macaque homologs of Kaposi's sarcoma-associated herpesvirus (KSHV) infect germinal center lymphoid cells, epithelial cells in skin and gastrointestinal tract and gonadal germ cells in naturally infected macaques.

We developed a set of rabbit antisera to characterize infections by the macaque RV2 rhadinovirus homologs of KSHV. We analyzed tissues from rhesus and pig-tailed macaques naturally infected with rhesus rhadinovirus (RRV) or Macaca nemestrina rhadinovirus 2 (MneRV2). Our study demonstrates that RV2 rhadinoviruses have a tropism for epithelial cells, lymphocytes and gonadal germ cells in vivo. We observed latent infections in both undifferentiated and differentiated epithelial cells with expression of the latency marker, LANA. Expression of the early (ORF59) and late (glycoprotein B) lytic markers were detected in highly differentiated cells in epithelial ducts in oral, renal, dermal and gastric mucosal tissue as well as differentiated germ cells in male and female gonads. Our data provides evidence that epithelial and germ cell differentiation in vivo induces rhadinovirus reactivation and suggests that infected epithelial and germ cells play a role in transmission and dissemination of RV2 rhadinovirus infections in vivo.

Risk of Contamination of Gametes and Embryos during Cryopreservation and Measures to Prevent Cross-Contamination.

The introduction and widespread application of vitrification are one of the most important achievements in human assisted reproduction techniques (ART) of the past decade despite controversy and unclarified issues, mostly related to concerns about disease transmission. Guidance documents published by US Food and Drug Administration, which focused on the safety of tissue/organ donations during Zika virus spread in 2016, as well as some reports of virus, bacteria, and fungi survival to cryogenic temperatures, highlighted the need for a review of the way how potentially infectious material is handled and stored in ART-related procedures. It was experimentally demonstrated that cross-contamination between liquid nitrogen (LN2) and embryos may occur when infectious agents are present in LN2 and oocytes/embryos are not protected by a hermetically sealed device. Thus, this review summarizes pertinent data and opinions regarding the potential hazard of infectious transmission through cryopreserved and banked reproductive cells and tissues in LN2. Special attention is given to the survival of pathogens in LN2, the risk of cross-contamination, vitrification methods, sterility of LN2, and the risks associated with the use of straws, cryovials, and storage dewars.

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling.

BACKGROUND: Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ∼1% of the population. OBJECTIVES: Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition. STUDY DESIGN: A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor-recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome. RESULTS: Thirty-one pools had high positive HHV-6 DNA detection with >10(3) HHV-6 DNA copies/µg. Each pool had one sample with >10(4) copies/µg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>10(3) HHV-6 DNA copies/µg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing. CONCLUSIONS: Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact.

Proliferation of endogenous retroviruses in the early stages of a host germ line invasion.

Endogenous retroviruses (ERVs) comprise 8% of the human genome and are common in all vertebrate genomes. The only retrovirus known to be currently transitioning from exogenous to endogenous form is the koala retrovirus (KoRV), making koalas (Phascolarctos cinereus) ideal for examining the early stages of retroviral endogenization. To distinguish endogenous from exogenous KoRV proviruses, we isolated koala genomic regions flanking KoRV integration sites. In three wild southern Australian koalas, there were fewer KoRV loci than in three captive Queensland koalas, consistent with reports that southern Australian koalas carry fewer KoRVs. Of 39 distinct KoRV proviral loci examined in a sire-dam-progeny triad, all proved to be vertically transmitted and endogenous; none was exogenous. Of the 39 endogenous KoRVs (enKoRVs), only one was present in the genomes of both the sire and the dam, suggesting that, at this early stage in the retroviral invasion of a host germ line, very large numbers of ERVs have proliferated at very low frequencies in the koala population. Sequence divergence between the 5'- and 3'-long terminal repeats (LTRs) of a provirus can be used as a molecular clock. Within each of ten enKoRVs, the 5'-LTR sequence was identical to the 3'-LTR sequence, suggesting a maximum age for enKoRV invasion of the koala germ line of approximately 22,200-49,900 years ago, although a much younger age is possible. Across the ten proviruses, seven LTR haplotypes were detected, indicating that at least seven different retroviral sequences had entered the koala germ line.

Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators.

Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.

Germ-line transmitted, chromosomally integrated HHV-6 and classical Hodgkin lymphoma.

A unique feature of both human herpesvirus 6A and B (HHV-6A and B) among human herpesviruses is their ability to integrate into chromosomal telomeres. In some individuals integrated viral genomes are present in the germ-line and result in the vertical transmission of HHV-6; however, little is known about the disease associations of germ-line transmitted, chromosomally integrated HHV-6 (ciHHV-6). Recent publications suggest that HHV-6 is associated with classical Hodgkin lymphoma (cHL). Here we examine the prevalence of ciHHV-6 in 936 cases of cHL and 563 controls by screening with a duplex TaqMan assay and confirming with droplet digital PCR. ciHHV-6 was detected in 10/563 (1.8%) controls and in all but one individual the virus was HHV-6B. Amongst cases 16/936 (1.7%) harboured ciHHV-6, thus demonstrating no association between ciHHV-6 and risk of cHL.