Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Adaptation of CD4 in gorillas and chimpanzees conveyed resistance to simian immunodeficiency viruses.

Simian immunodeficiency viruses (SIVs) comprise a large group of primate lentiviruses that endemically infect African monkeys. HIV-1 spilled over to humans from this viral reservoir, but the spillover did not occur directly from monkeys to humans. Instead, a key event was the introduction of SIVs into great apes, which then set the stage for infection of humans. Here, we investigate the role of the lentiviral entry receptor, CD4, in this key and fateful event in the history of SIV/HIV emergence. First, we reconstructed and tested ancient forms of CD4 at two important nodes in ape speciation, both prior to the infection of chimpanzees and gorillas with these viruses. These ancestral CD4s fully supported entry of diverse SIV isolates related to the viruses that made this initial jump to apes. In stark contrast, modern chimpanzee and gorilla CD4 orthologs are more resistant to these viruses. To investigate how this resistance in CD4 was gained, we acquired CD4 gene sequences from 32 gorilla individuals of two species, and identified alleles that encode 8 unique CD4 protein variants. Functional testing of these identified variant-specific differences in susceptibility to virus entry. By engineering single point mutations from resistant gorilla CD4 variants into the permissive human CD4 receptor, we demonstrate that acquired substitutions in gorilla CD4 did convey resistance to virus entry. We provide a population genetic analysis to support the theory that selection is acting in favor of more and more resistant CD4 alleles in ape species harboring SIV endemically (gorillas and chimpanzees), but not in other ape species that lack SIV infections (bonobos and orangutans). Taken together, our results show that SIV has placed intense selective pressure on ape CD4, acting to propagate SIV-resistant alleles in chimpanzee and gorilla populations.

Human mRNA in saliva can correctly identify individuals harboring acute infection.

The COVID-19 pandemic demonstrated the poor ability of body temperature to reliably identify SARS-CoV-2-infected individuals, an observation that has been made before in the context of other infectious diseases. While acute infection does not always cause fever, it does reliably drive host transcriptional responses as the body responds at the site of infection. These transcriptional changes can occur both in cells that are directly harboring replicating pathogens and in cells elsewhere that receive a molecular signal that infection is occurring. Here, we identify a core set of approximately 70 human genes that are together upregulated in cultured human cells infected by a broad array of viral, bacterial, and fungal pathogens. We have named these "core response" genes. In theory, transcripts from these genes could serve as biomarkers of infection in the human body, in a way that is agnostic to the specific pathogen causing infection. As such, we perform human studies to show that these infection-induced human transcripts can be measured in the saliva of people harboring different types of infections. The number of these transcripts in saliva can correctly classify infection status (whether a person harbors an infection) 91% of the time. Furthermore, in the case of SARS-CoV-2 specifically, the number of core response transcripts in saliva correctly identifies infectious individuals even when enrollees, themselves, are asymptomatic and do not know they are infected.IMPORTANCEThere are a variety of clinical and laboratory criteria available to clinicians in controlled healthcare settings to help them identify whether an infectious disease is present. However, in situations such as a new epidemic caused by an unknown infectious agent, in health screening contexts performed within communities and outside of healthcare facilities or in battlefield or potential biowarfare situations, this gets more difficult. Pathogen-agnostic methods for rapid screening and triage of large numbers of people for infection status are needed, in particular methods that might work on an easily accessible biospecimen like saliva. Here, we identify a small, core set of approximately 70 human genes whose transcripts serve as saliva-based biomarkers of infection in the human body, in a way that is agnostic to the specific pathogen causing infection.

Regulation of human interferon signaling by transposon exonization.

Innate immune signaling is essential for clearing pathogens and damaged cells, and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues, and functions as a decoy receptor that potently inhibits interferon signaling including in cells infected with SARS-CoV-2. Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation.

Quantification of virus-infected cells using RNA FISH-Flow.

We present a protocol to detect cells that have been infected by RNA viruses. The method, RNA fluorescence in situ hybridization flow cytometry (RNA FISH-Flow), uses 48 fluorescently labeled DNA probes that hybridize in tandem to viral RNA. RNA FISH-Flow probes can be synthesized to match any RNA virus genome, in either sense or anti-sense, enabling detection of genomes or replication intermediates within cells. Flow cytometry enables high-throughput analysis of infection dynamics within a population at the single cell level. For complete details on the use and execution of this protocol, please refer to Warren et al. (2022).1.

How avian influenza viruses spill over to mammals.

The H3N2 canine influenza virus - which originally came from birds - is evolving to become more transmissible between dogs.

Structurally divergent and recurrently mutated regions of primate genomes.

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

Identifying animal viruses in humans.

Experimental virology can inform strategic monitoring for new viruses in humans.

High rates of observed face mask use at Colorado universities align with students' opinions about masking and support the safety and viability of in-person higher education during the COVID-19 pandemic.

BACKGROUND: Over the course of the COVID-19 pandemic, colleges and universities have focused on creating policies, such as mask mandates, to minimize COVID-19 transmission both on their campuses and in the surrounding community. Adherence to and opinions about these policies remain largely unknown. METHODS: The Centers for Disease Control and Prevention (CDC) developed a cross-sectional study, the Mask Adherence and Surveillance at Colleges and Universities Project (MASCUP!), to objectively and inconspicuously measure rates of mask use at institutes of higher education via direct observation. From February 15 through April 11, 2021 the University of Colorado Boulder (CU, n = 2,808 observations) and Colorado State University Fort Collins (CSU, n = 3,225 observations) participated in MASCUP! along with 52 other institutes of higher education (n = 100,353 observations) spanning 21 states and the District of Columbia. Mask use was mandatory at both Colorado universities and student surveys were administered to assess student beliefs and attitudes. RESULTS: We found that 91.7%, 93.4%, and 90.8% of persons observed at indoor locations on campus wore a mask correctly at University of Colorado, Colorado State University, and across the 52 other schools, respectively. Student responses to questions about masking were in line with these observed rates of mask use where 92.9% of respondents at CU and 89.8% at CSU believe that wearing masks can protect the health of others. Both Colorado universities saw their largest surges in COVID-19 cases in the fall of 2020, with markedly lower case counts during the mask observation window in the spring of 2021. CONCLUSION: High levels of mask use at Colorado's two largest campuses aligned with rates observed at other institutes across the country. These high rates of use, coupled with positive student attitudes about mask use, demonstrate that masks were widely accepted and may have contributed to reduced COVID-19 case counts. This study supports an emerging body of literature substantiating masks as an effective, low-cost measure to reduce disease transmission and establishes masking (with proper education and promotion) as a viable tactic to reduce respiratory disease transmission on college campuses.

RNase L activation in the cytoplasm induces aberrant processing of mRNAs in the nucleus.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.

Primate hemorrhagic fever-causing arteriviruses are poised for spillover to humans.

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

Monkeypox emergency: Urgent questions and perspectives.

Amidst the COVID-19 pandemic, we now face another public health emergency in the form of monkeypox virus. As of August 1, the Centers for Disease Control and Prevention report over 23,000 cases in 80 countries. An inclusive and global collaborative effort to understand the biology, evolution, and spread of the virus as well as commitment to vaccine equity will be critical toward containing this outbreak. We share the voices of leading experts in this space on what they see as the most pressing questions and directions for the community.

Human ACE2 Polymorphisms from Different Human Populations Modulate SARS-CoV-2 Infection.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 6 million deaths worldwide. The high variability in COVID-19 symptoms remains one of the most interesting mysteries of the pandemic. Genetic and environmental factors are likely to be key determinants of COVID-19 symptomatology. Here, we explored ACE2 as a genetic determinant for SARS-CoV-2 infection and COVID-19 symptomatology. Each human genome encodes two alleles of ACE2, which encodes the cell entry receptor for SARS-CoV-2. Here, we determined whether naturally occurring human ACE2 (hACE2) polymorphisms in the human population affect SARS-CoV-2 infection and the severity of COVID-19 symptoms. ACE2 variants S19P, I21V, E23K, K26R, K31R, N33I, H34R, E35K, and T92I showed increased virus infection compared to wild-type ACE2; thus, these variants could increase the risk for COVID-19. In contrast, variants D38V, Y83H, I468V, and N638S showed reduced infection, indicating a potential protective effect. hACE2 variants K26R and T92I increased infection by three-fold without changing the levels of ACE2 on the surface of the cells, suggesting that these variants may increase the risk of severe COVID-19. On the contrary, hACE2 variants D38V and Y83H decreased SARS-CoV-2 infection by four- and ten-fold, respectively, without changing surface expression, suggesting that these variants may protect against severe COVID-19. Remarkably, all protective hACE2 Polymorphisms were found almost exclusively in Asian populations, which may provide a partial explanation for the low COVID-19 mortality rates in Asian countries. Thus, hACE2 polymorphisms may modulate susceptibility to SARS-CoV-2 in the host and partially account for the differences in severity of COVID-19 among different ethnic groups.

Nuku, a family of primate retrocopies derived from KU70.

The gene encoding the ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived "NUKU" retrocopies reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrocopies show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible tissue-specific translation. Molecular modeling predicted that amino acid changes in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrocopies appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrocopy is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retrovirus transduction in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology.

RNase L limits host and viral protein synthesis via inhibition of mRNA export.

RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L-mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L-mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.

Positive natural selection in primate genes of the type I interferon response.

BACKGROUND: The Type I interferon response is an important first-line defense against viruses. In turn, viruses antagonize (i.e., degrade, mis-localize, etc.) many proteins in interferon pathways. Thus, hosts and viruses are locked in an evolutionary arms race for dominance of the Type I interferon pathway. As a result, many genes in interferon pathways have experienced positive natural selection in favor of new allelic forms that can better recognize viruses or escape viral antagonists. Here, we performed a holistic analysis of selective pressures acting on genes in the Type I interferon family. We initially hypothesized that the genes responsible for inducing the production of interferon would be antagonized more heavily by viruses than genes that are turned on as a result of interferon. Our logic was that viruses would have greater effect if they worked upstream of the production of interferon molecules because, once interferon is produced, hundreds of interferon-stimulated proteins would activate and the virus would need to counteract them one-by-one. RESULTS: We curated multiple sequence alignments of primate orthologs for 131 genes active in interferon production and signaling (herein, "induction" genes), 100 interferon-stimulated genes, and 100 randomly chosen genes. We analyzed each multiple sequence alignment for the signatures of recurrent positive selection. Counter to our hypothesis, we found the interferon-stimulated genes, and not interferon induction genes, are evolving significantly more rapidly than a random set of genes. Interferon induction genes evolve in a way that is indistinguishable from a matched set of random genes (22% and 18% of genes bear signatures of positive selection, respectively). In contrast, interferon-stimulated genes evolve differently, with 33% of genes evolving under positive selection and containing a significantly higher fraction of codons that have experienced selection for recurrent replacement of the encoded amino acid. CONCLUSION: Viruses may antagonize individual products of the interferon response more often than trying to neutralize the system altogether.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the Fall 2020 pandemic response efforts at the University of Colorado Boulder (USA), where more than 72,500 saliva samples were tested for SARS-CoV-2 using quantitative RT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously reported in symptomatic individuals. Regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in non-infectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "super-carriers" and possibly also super-spreaders.

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.