Biology subjects

Pung, L. J.

Publications and source records attributed to Pung, L. J..

3 recordsLinked to original sources

bioRxiv2025

Infection Of Rhesus Macaques With Onyong-nyong Virus UVIR-O804 Recapitulates Key Aspects of Human Clinical Disease

1.Onyong-nyong virus (ONNV) is a mosquito-borne alphavirus first isolated in Uganda in 1959. Since its discovery, ONNV has caused several outbreaks in Africa, manifesting clinically as fever, rash, and joint/muscle pain lasting months. Currently, we have a limited understanding of ONNV infection and disease in relevant animal models, which restricts the evaluation of vaccines and therapeutics. In 1967, Binn et al. reported that infection of rhesus macaques (RMs) with ONNV failed to induce viremia in two animals. This may be attributed to the potential attenuation of the virus through extensive passaging. To mitigate this issue, we constructed an infectious clone from the sequence of ONNV-UVRI0804 (ONNV0804), a 2017 clinical isolate from a febrile patient in Uganda. This strain demonstrated high pathogenicity in immunocompetent mice, resulting in an earlier and more severe onset of disease and significantly higher viremia compared to a highly passaged control strain ONNVUgMP30. In the current study, three male and three female rhesus macaques were subcutaneously inoculated with ONNV0804. All animals became viremic at 2 days post inoculation (dpi). Both classical and nonclassical monocytes were activated (CD169+), peaking at 3 dpi, which corresponded with the peak of viremia. Additionally, CD4+ and CD8+ effector memory T cells and memory B cells began proliferating in peripheral blood by day 7, peaking at day 10, which also corresponded to the timing of neutralizing antibody development, indicating a robust adaptive immune response to ONNV0804. Finally, key clinical disease manifestations were recapitulated, including lymphadenopathy and histological features of early-stage arthritis. Taken together, rhesus macaque infection with ONNV0804 clinical isolate is a promising model for investigating immune responses to alphaviruses and evaluating vaccines to protect against future epidemics. 2. Author summaryOnyong-nyong virus (ONNV) is a mosquito-transmitted alphavirus that causes fever, rash, and prolonged joint and muscle pain, similar to chikungunya virus and other arthritogenic alphaviruses. Despite its capacity to cause outbreaks in Africa, ONNV remains understudied, and there are currently no approved vaccines or therapeutics to prevent or treat infection and disease. A major barrier to advancing ONNV research has been the lack of suitable animal models to study the virus and investigate host immune responses. We engineered an ONNV infectious clone of a recent clinical isolate sequenced from a patient in Uganda (ONNV0804) that causes robust infection and disease in immunocompetent mice. In the current study, we provide data demonstrating that this contemporary ONNV strain infects rhesus macaques. Notably, rhesus macaques developed detectable viremia, rash, lymphadenopathy, joint and muscle inflammation, and strong innate and adaptive immune responses following subcutaneous ONNV0804 infection. These findings suggest that ONNV0804 infection in macaques is a promising model for studying ONNV pathogenesis and immunity. This model will be instrumental for evaluating future vaccine and therapeutic candidates aimed at preventing ONNV infection and related viral disease.

microbiology↗

bioRxiv2025

A Zika Virus-Like Particle Vaccine Mitigates Early Pregnancy Loss In Rhesus Macaques

Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in the Americas in 2015-16. In Brazil, up to 46% of ZIKV positive pregnancies resulted in congenital Zika syndrome (CZS). CZS is characterized by a wide range of neurologic birth defects and miscarriage in up to 7.6% of affected pregnancies. With no current licensed ZIKV vaccines, we sought to evaluate a Zika virus-like particle (VLP) vaccine candidate in a rhesus macaque (RM) pregnancy model. VLPs were produced in mammalian cells expressing the pre-membrane-envelope region of the Asian-lineage ZIKV strain PRVABC59, which belongs to the Asian ZIKV lineage that is associated with outbreaks of congenital disease. To evaluate vaccine protection against adverse pregnancy complications, two cohorts of female RM were vaccinated with ZIKV-VLP with adjuvant Alhydrogel (alum) or adjuvant alone prior to mating. At gestational day (GD) 30 (early first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain shown to induce 1st-trimester fetal demise in 78% (n=11/14 animals) of RM, making it an ideal and stringent model for evaluating ZIKV vaccines. Within the vaccinated cohort, 2 of 3 animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. The third animal experienced pregnancy loss at GD 49 (18 d post infection), although no infectious virus was detected in placental or fetal tissues. In the unvaccinated cohort, two animals had severe adverse events. One animal experienced preterm labor, and another developed early-onset hydrops fetalis with widespread ZIKV-RNA detected via RNAscope and extensive placental damage. These results confirm a significant risk for early pregnancy loss in RM infected with ZIKV-DAK 41524. This model can be further used to understand the complexities of placental immunological features underlying stillbirth and miscarriage following infection. Our findings indicate that this ZIKV-VLP vaccine candidate protected pregnant macaques against fetal demise associated with highly pathogenic ZIKV challenge. Author SummaryZika virus (ZIKV) infection during pregnancy is associated with pregnancy loss, severe birth defects, including microcephaly and developmental delays, and other subtle neurologic changes. Although vaccine development efforts have been ongoing since the 2015/2016 ZIKV outbreak, no approved vaccine is currently available. Many existing studies have tested vaccines in animal models using strains such as ZIKV-PR that cause mild or moderate pregnancy complications at similar rates to human cases. Using challenge strains that only cause mild, or moderate pregnancy complications makes it difficult to rigorously evaluate vaccine efficacy. In this study, we tested a virus-like particle (VLP) vaccine, a safe and effective method for use during pregnancy as it is replication-deficient and only contains viral structural antigens. We found that the VLP vaccine, when paired with an adjuvant (alum), induced strong antibody responses in mice and controlled viral dissemination following challenge in nonpregnant macaques. To evaluate the protective efficacy of the ZIKV-VLP vaccine against in utero infection and disease, we used a stringent model of ZIKV infection during early pregnancy in rhesus macaques that is associated with high rates of fetal demise. In pregnant macaques, the vaccine reduced maternal viremia, limited viral spread to fetal and placental tissues, and conferred protection against virus-mediated fetal demise and placental damage. This is the first study to evaluate a VLP-based vaccine in a consistent pregnancy loss model of ZIKV infection. These findings support the continued development of VLP vaccines as a safe and effective strategy for protecting pregnant individuals and their developing fetuses from ZIKV.

microbiology↗

bioRxiv2025

Human Cytomegalovirus UL78 is a Nuclear-Localized GPCR Necessary for Efficient Reactivation from Latent Infection in CD34+ Hematopoietic Progenitor Cells

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that persists throughout the lifetime of the host due in part to the establishment of latency in CD34+ hematopoietic progenitor cells (HPCs) and CD14+ monocytes. HCMV encodes four putative G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. While the roles of most of these receptors have been investigated, a definitive role for UL78 in HCMV infection has yet to be elucidated. Utilizing an in vitro CD34+ HPC model, we demonstrate that a recombinant virus lacking UL78 protein expression fails to efficiently reactivate from latent infection. Furthermore, we show using a Lumit-based assay that UL78 preferentially couples to the Gi family of G proteins and that a recombinant HCMV containing mutations in the UL78 G protein-coupling DRL motif also fails to reactivate from latent infection. Together our findings indicate that Gi coupling is important for UL78 function during reactivation in latently infected CD34+ HPCs, however the protein is not required to establish or maintain latency. To better understand the role of UL78, we conducted proximity-dependent labeling analyses in HCMV-UL78-TurboID infected fibroblasts and CD34+ HPCs undergoing reactivation from latency. Congruent with our coupling data, we found Gi was the only heterotrimeric G protein in proximity to UL78. Pathway analysis of the UL78 interactome revealed proteins associated with membrane trafficking, signaling, and the nuclear pore complex as enriched in both cell types. In addition, the UL78 interactome contained viral proteins with nuclear localization including viral transcription and DNA replication machinery. Nuclear localization of UL78 was validated using cell fractionation, immunofluorescence microscopy, and proximity-dependent labelling of isolated nuclei. Together, our results provide novel insights into the localization and function of UL78, previously unknown to contribute to reactivation from latent infection. Author SummaryHuman cytomegalovirus (HCMV) remains one of the most widespread viral infections globally. Primary HCMV infection is typically asymptomatic and leads to the establishment of latency in myeloid lineage cells, where the virus persists for the hosts lifetime. Reactivation of latent HCMV can cause severe complications, particularly in immunocompromised individuals such as transplant recipients and people living with HIV. Several factors influence the transition from latent to lytic infection, including signal transduction through the viral G protein-coupled receptors: US27, US28, UL33, and UL78. Using an advanced in vitro model, we show that recombinant viruses lacking UL78 fail to efficiently reactivate from latent infection. Moreover, we show that UL78 preferentially couples to the Gi family of G proteins via a conserved DRL motif, and this coupling is required for efficient reactivation. These results were confirmed by proximity-dependent labeling experiments where we identified Gi and several other proteins involved in trafficking, signaling, transcription, and nuclear localization. Nuclear localization of UL78 was confirmed by cell fractionation, immunofluorescence microscopy, and proximity-dependent labeling in isolated nuclei. Collectively, our results uncover a novel role for UL78 in reactivation from latency and shed new light on its localization and function.

microbiology↗