Forging Collaborative Relationships in Brazil: From AIDS to ZIKV.

The Zika virus (ZIKV) outbreak has stimulated collaborations between Brazilians, researchers from other South American countries, and scientists from around the world. The Brazilian response to the HIV/AIDS epidemic demonstrates capabilities that can be applied to the study of ZIKV and provides lessons for developing effective international infectious disease research collaborations.

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Complete sequencing of a cynomolgus macaque major histocompatibility complex haplotype.

Macaques provide the most widely used nonhuman primate models for studying the immunology and pathogenesis of human diseases. Although the macaque major histocompatibility complex (MHC) region shares most features with the human leukocyte antigen (HLA) region, macaques have an expanded repertoire of MHC class I genes. Although a chimera of two rhesus macaque MHC haplotypes was first published in 2004, the structural diversity of MHC genomic organization in macaques remains poorly understood owing to a lack of adequate genomic reference sequences. We used ultralong Oxford Nanopore and high-accuracy Pacific Biosciences (PacBio) HiFi sequences to fully assemble the ∼5.2-Mb M3 haplotype of an MHC-homozygous, Mauritian-origin cynomolgus macaque (Macaca fascicularis). The MHC homozygosity allowed us to assemble a single MHC haplotype unambiguously and avoid chimeric assemblies that hampered previous efforts to characterize this exceptionally complex genomic region in macaques. The high quality of this new assembly is exemplified by the identification of an extended cluster of six Mafa-AG genes that contains a recent duplication with a highly similar ∼48.5-kb block of sequence. The MHC class II region of this M3 haplotype is similar to the previously sequenced rhesus macaque haplotype and HLA class II haplotypes. The MHC class I region, in contrast, contains 13 MHC-B genes, four MHC-A genes, and three MHC-E genes (vs. 19 MHC-B, two MHC-A, and one MHC-E in the previously sequenced haplotype). These results provide an unambiguously assembled single contiguous cynomolgus macaque MHC haplotype with fully curated gene annotations that will inform infectious disease and transplantation research.

Determinants of pegivirus persistence, cross-species infection, and adaptation in the laboratory mouse.

Viruses capable of causing persistent infection have developed sophisticated mechanisms for evading host immunity, and understanding these processes can reveal novel features of the host immune system. One such virus, human pegivirus (HPgV), infects ~15% of the global human population, but little is known about its biology beyond the fact that it does not cause overt disease. We passaged a pegivirus isolate of feral brown rats (RPgV) in immunodeficient laboratory mice to develop a mouse-adapted virus (maPgV) that established persistent high-titer infection in a majority of wild-type laboratory mice. maRPgV viremia was detected in the blood of mice for >300 days without apparent disease, closely recapitulating the hallmarks of HPgV infection in humans. We found a pro-viral role for type-I interferon in chronic infection; a lack of PD-1-mediated tolerance to PgV infection; and multiple mechanisms by which PgV immunity can be achieved by an immunocompetent host. These data indicate that the PgV immune evasion strategy has aspects that are both common and unique among persistent viral infections. The creation of maPgV represents the first PgV infection model in wild-type mice, thus opening the entire toolkit of the mouse host to enable further investigation of this persistent RNA virus infections.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

Complete Genomic Assembly of Mauritian Cynomolgus Macaque Killer Ig-like Receptor and Natural Killer Group 2 Haplotypes.

Mauritian-origin cynomolgus macaques (MCMs) serve as a powerful nonhuman primate model in biomedical research due to their unique genetic homogeneity, which simplifies experimental designs. Despite their extensive use, a comprehensive understanding of crucial immune-regulating gene families, particularly killer Ig-like receptors (KIR) and NK group 2 (NKG2), has been hindered by the lack of detailed genomic reference assemblies. In this study, we employ advanced long-read sequencing techniques to completely assemble eight KIR and seven NKG2 genomic haplotypes, providing an extensive insight into the structural and allelic diversity of these immunoregulatory gene clusters. Leveraging these genomic resources, we prototype a strategy for genotyping KIR and NKG2 using short-read, whole-exome capture data, illustrating the potential for cost-effective multilocus genotyping at colony scale. These results mark a significant enhancement for biomedical research in MCMs and underscore the feasibility of broad-scale genetic investigations.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Frequent first-trimester pregnancy loss in rhesus macaques infected with African-lineage Zika virus.

In the 2016 Zika virus (ZIKV) pandemic, a previously unrecognized risk of birth defects surfaced in babies whose mothers were infected with Asian-lineage ZIKV during pregnancy. Less is known about the impacts of gestational African-lineage ZIKV infections. Given high human immunodeficiency virus (HIV) burdens in regions where African-lineage ZIKV circulates, we evaluated whether pregnant rhesus macaques infected with simian immunodeficiency virus (SIV) have a higher risk of African-lineage ZIKV-associated birth defects. Remarkably, in both SIV+ and SIV- animals, ZIKV infection early in the first trimester caused a high incidence (78%) of spontaneous pregnancy loss within 20 days. These findings suggest a significant risk for early pregnancy loss associated with African-lineage ZIKV infection and provide the first consistent ZIKV-associated phenotype in macaques for testing medical countermeasures.

Isolation of Diverse Simian Arteriviruses Causing Hemorrhagic Disease.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Shedding of infectious SARS-CoV-2 despite vaccination.

The SARS-CoV-2 Delta Variant of Concern is highly transmissible and contains mutations that confer partial immune escape. The emergence of Delta in North America caused the first surge in COVID-19 cases after SARS-CoV-2 vaccines became widely available. To determine whether individuals infected despite vaccination might be capable of transmitting SARS-CoV-2, we compared RT-PCR cycle threshold (Ct) data from 20,431 test-positive anterior nasal swab specimens from fully vaccinated (n = 9,347) or unvaccinated (n = 11,084) individuals tested at a single commercial laboratory during the interval 28 June- 1 December 2021 when Delta variants were predominant. We observed no significant effect of vaccine status alone on Ct value, nor when controlling for vaccine product or sex. Testing a subset of low-Ct (<25) samples, we detected infectious virus at similar rates, and at similar titers, in specimens from vaccinated and unvaccinated individuals. These data indicate that vaccinated individuals infected with Delta variants are capable of shedding infectious SARS-CoV-2 and could play a role in spreading COVID-19.

A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

The landscape of antibody binding in SARS-CoV-2 infection.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Novel Lymphocytic Choriomeningitis Virus Strain Sustains Abundant Exhausted Progenitor CD8 T Cells without Systemic Viremia.

Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus and a natural mouse pathogen. LCMV-Armstrong, an acutely resolved strain, and LCMV-clone 13, a mutant that establishes chronic infection, have provided contrasting infection models that continue to inform the fundamental biology of T cell differentiation, regulation of exhaustion, and response to checkpoint blockade. In this study, we report the isolation and characterization of LCMV-Minnesota (LCMV-MN), which was naturally transmitted to laboratory mice upon cohousing with pet shop mice and shares 80-95% amino acid homology with previously characterized LCMV strains. Infection of laboratory mice with purified LCMV-MN resulted in viral persistence that was intermediate between LCMV-Armstrong and -clone 13, with widely disseminated viral replication and viremia that was controlled within 15-30 d, unless CD4 T cells were depleted prior to infection. LCMV-MN-responding CD8+ T cells biased differentiation toward the recently described programmed death-1 (PD-1)+CXCR5+Tim-3lo stemlike CD8+ T cell population (also referred to as progenitor exhausted T cells) that effectuates responses to PD-1 blockade checkpoint inhibition, a therapy that rejuvenates responses against chronic infections and cancer. This subset resembled previously characterized PD-1+TCF1+ stemlike CD8+ T cells by transcriptional, phenotypic, and functional assays, yet was atypically abundant. LCMV-MN may provide a tool to better understand the breadth of immune responses in different settings of chronic Ag stimulation as well as the ontogeny of progenitor exhausted T cells and the regulation of responsiveness to PD-1 blockade.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Acute SARS-CoV-2 infections harbor limited within-host diversity and transmit via tight transmission bottlenecks.

The emergence of divergent SARS-CoV-2 lineages has raised concern that novel variants eliciting immune escape or the ability to displace circulating lineages could emerge within individual hosts. Though growing evidence suggests that novel variants arise during prolonged infections, most infections are acute. Understanding how efficiently variants emerge and transmit among acutely-infected hosts is therefore critical for predicting the pace of long-term SARS-CoV-2 evolution. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely-infected individuals. We find that within-host diversity is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household, and are rarely detected along phylogenetically linked infections in the broader community. These findings suggest that most variation generated within-host is lost during transmission.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Sensitivity of rapid antigen tests for Omicron subvariants of SARS-CoV-2.

Diagnosis by rapid antigen tests (RATs) is useful for early initiation of antiviral treatment. Because RATs are easy to use, they can be adapted for self-testing. Several kinds of RATs approved for such use by the Japanese regulatory authority are available from drug stores and websites. Most RATs for COVID-19 are based on antibody detection of the SARS-CoV-2 N protein. Since Omicron and its subvariants have accumulated several amino acid substitutions in the N protein, such amino acid changes might affect the sensitivity of RATs. Here, we investigated the sensitivity of seven RATs available in Japan, six of which are approved for public use and one of which is approved for clinical use, for the detection of BA.5, BA.2.75, BF.7, XBB.1, and BQ.1.1, as well as the delta variant (B.1.627.2). All tested RATs detected the delta variant with a detection level between 7500 and 75 000 pfu per test, and all tested RATs showed similar sensitivity to the Omicron variant and its subvariants (BA.5, BA.2.75, BF.7, XBB.1, and BQ.1.1). Human saliva did not reduce the sensitivity of the RATs tested. Espline SARS-CoV-2 N showed the highest sensitivity followed by Inspecter KOWA SARS-CoV-2 and V Trust SARS-CoV-2 Ag. Since the RATs failed to detect low levels of infectious virus, individuals whose specimens contained less infectious virus than the detection limit would be considered negative. Therefore, it is important to note that RATs may miss individuals shedding low levels of infectious virus.

A new genotype of hepatitis A virus causing transient liver enzyme elevations in Mauritius-origin laboratory-housed Macaca fascicularis.

Hepatitis A virus (HAV) infects humans and nonhuman primates, typically causing an acute self-limited illness. Three HAV genotypes have been described so far for humans, and three genotypes have been described for nonhuman primates. We observed transiently elevated liver enzymes in Mauritius-origin laboratory-housed macaques in Germany and were not able to demonstrate an etiology including HAV by serology and polymerase chain reaction (PCR). HAV is a rare pathogen in cynomolgus macaques, and since all employees were routinely vaccinated against HAV, it was not a part of the routine vaccination and screening program. A deep sequencing approach identified a new HAV genotype (referred to as Simian_HAV_Macaca/Germany/Mue-1/2022) in blood samples from affected animals. This HAV was demonstrated by reverse transcription PCR in blood and liver and by in situ hybridization in liver, gall bladder, and septal ducts. A commercial vaccine was used to protect animals from liver enzyme elevation. The newly identified simian HAV genotype demonstrates 80% nucleotide sequence identity to other simian and human HAV genotypes. There was deeper divergence between Simian_HAV_Macaca/Germany/Mue-1/2022 and other previously described HAVs, including both human and simian viruses. In situ hybridization indicated persistence in the biliary epithelium up to 3 months after liver enzymes were elevated. Vaccination using a commercial vaccine against human HAV prevented reoccurrence of liver enzyme elevations. Because available assays for HAV did not detect this new HAV genotype, knowledge of its existence may ameliorate potential significant epidemiological and research implications in laboratories globally.