Dawn M. Dudley

A rhesus macaque model of Asian-lineage Zika virus infection

Infection with Asian-lineage Zika virus (ZIKV) has been associated with Guillain–Barré syndrome and fetal abnormalities, but the underlying mechanisms remain poorly understood. Animal models of infection are thus urgently needed. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage ZIKV closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, ZIKV RNA is detected in plasma 1 day post infection (d.p.i.) in all animals (N=8, including 2 pregnant animals), and is also present in saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 days and for up to at least 57 days, respectively. Neutralizing antibodies are detected by 21 d.p.i. Rechallenge 10 weeks after the initial challenge results in no detectable virus replication, indicating protective immunity against homologous strains. Therefore, Asian-lineage ZIKV infection of rhesus macaques provides a relevant animal model for studying pathogenesis and evaluating potential interventions against human infection, including during pregnancy.

Whole-genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep pyrosequencing.

ABSTRACT Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

Low-cost ultra-wide genotyping using Roche/454 pyrosequencing for surveillance of HIV drug resistance.

Background Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10–12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques

Background Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV. Methodology/Principal Findings Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form. Conclusions/Significance An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.

Selection of a Simian-Human Immunodeficiency Virus Strain Resistant to a Vaginal Microbicide in Macaques

ABSTRACT PSC-RANTES binds to CCR5, inhibits human immunodeficiency virus type 1 (HIV-1) entry, and has been shown as a vaginal microbicide to protect rhesus macaques from a simian-human immunodeficiency virus chimera (SHIVSF162-p3) infection in a dose-dependent manner. In this study, env gene sequences from SHIVSF162-p3-infected rhesus macaques treated with PSC-RANTES were analyzed for possible drug escape variants. Two specific mutations located in the V3 region of gp120 (K315R) and C-helical domain of gp41 (N640D) were identified in a macaque (m584) pretreated with a 100 μM dose of PSC-RANTES. These two env mutations were found throughout infection (through week 77) but were found at only low frequencies in the inoculating SHIVSF162-p3 stock and in the other SHIVSF162-p3-infected macaques. HIV-1 env genes from macaque m584 (envm584) and from inoculating SHIVSF162-p3 (envp3) were cloned into an HIV-1 backbone. Increases in 50% inhibitory concentrations to PSC-RANTES with envm584 were modest (sevenfold) and most pronounced in cells expressing rhesus macaque CCR5 as compared to human CCR5. Nonetheless, virus harboring envm584, unlike inoculating virus envp3, could replicate even at the highest tissue culture PSC-RANTES concentrations (100 nM). Dual-virus competitions revealed a dramatic increase in fitness of chimeric virus containing envm584 (K315R/N640D) over that containing envp3, but again, only in rhesus CCR5-expressing cells. This study is the first to describe the immediate selection and infection of a drug-resistant SHIV variant in the face of a protective vaginal microbicide, PSC-RANTES. This rhesus CCR5-specific/PSC- RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIVSF162-p3 stock compared to the potential exposure to a heterogeneous HIV-1 population in human transmission.

Oropharyngeal mucosal transmission of Zika virus in rhesus macaques

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low.Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.

KIR3DL01 Recognition of Bw4 Ligands in the Rhesus Macaque: Maintenance of Bw4 Specificity since the Divergence of Apes and Old World Monkeys

The identification of MHC class I ligands for rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to our basic understanding of KIR and MHC class I coevolution and to the study of NK cell responses in this nonhuman primate model for AIDS and other viral diseases. In this study, we show that Mamu-KIR3DL01, which is expressed by ∼90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb which, in this study, was shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233. The cytolytic activity of Mamu-KIR3DL01+ NK cells was suppressed by cell lines expressing the Bw4 molecules Mamu-B*007:01, -B*041:01, -B*058:02, and -B*065:01. The Bw4 motif was necessary for Mamu-KIR3DL01 recognition because substitutions in this region abrogated Mamu-KIR3DL01+ NK cell inhibition. However, the presence of a Bw4 motif was not sufficient for recognition because another Bw4 molecule, Mamu-B*017:01, failed to suppress the cytolytic activity of these NK cells. Replacement of three residues in Mamu-B*017:01, predicted to be KIR contacts based on the three-dimensional structure of the human KIR3DL1-HLA-Bw4 complex, with the corresponding residues at these positions for the other Mamu-Bw4 ligands restored Mamu-KIR3DL01+ NK cell inhibition. These results define the ligand specificity of one of the most polymorphic and commonly expressed KIRs in the rhesus macaque and reveal similarities in Bw4 recognition by Mamu-KIR3DL01 and human KIR3DL1, despite the absence of an orthologous relationship between these two KIRs or conservation of surface residues predicted to interact with MHC class I ligands.

Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq

BackgroundViral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors.ResultsA universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method.ConclusionsHere we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.