Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins.

The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.

Biochemistry and Molecular Biology of Flaviviruses.

Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA-protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways.IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.

Roles of Pro-viral Host Factors in Mosquito-Borne Flavivirus Infections.

Identification and analysis of viral host factors is a growing area of research which aims to understand the how viruses molecularly interface with the host cell. Investigations into flavivirus-host interactions has led to new discoveries in viral and cell biology, and will potentially bolster strategies to control the important diseases caused by these pathogens. Here, we address the current knowledge of prominent host factors required for the flavivirus life-cycle and mechanisms by which they promote infection.

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation.

The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. IMPORTANCE: Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs.

Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon.

BACKGROUND: The identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas. METHODS/RESULTS: Based on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs. CONCLUSION: This discovery expands our knowledge of Mimiviridae evolution and ecology.

CD8 + T cell response promotes viral clearance and reduces chances of severe testicular damage in mouse models of long-term Zika virus infection of the testes.

Zika virus (ZIKV) causes human testicular inflammation and alterations in sperm parameters and causes testicular damage in mouse models. The involvement of individual immune cells in testicular damage is not fully understood. We detected virus in the testes of the interferon (IFN) α/ß receptor -/- A129 mice three weeks post-infection and found elevated chemokines in the testes, suggesting chronic inflammation and long-term infection play a role in testicular damage. In the testes, myeloid cells and CD4 + T cells were absent at 7 dpi but were present at 23 days post-infection (dpi), and CD8 + T cell infiltration started at 7 dpi. CD8 -/- mice with an antibody-depleted IFN response had a significant reduction in spermatogenesis, indicating that CD8 + T cells are essential to prevent testicular damage during long-term ZIKV infections. Our findings on the dynamics of testicular immune cells and importance of CD8 + T cells functions as a framework to understand mechanisms underlying observed inflammation and sperm alterations in humans.

Yellow Fever Emergence: Role of Heterologous Flavivirus Immunity in Preventing Urban Transmission.

During major, recent yellow fever (YF) epidemics in Brazil, human cases were attributed only to spillover infections from sylvatic transmission with no evidence of human amplification. Furthermore, the historic absence of YF in Asia, despite abundant peridomestic Aedes aegypti and naive human populations, represents a longstanding enigma. We tested the hypothesis that immunity from dengue (DENV) and Zika (ZIKV) flaviviruses limits YF virus (YFV) viremia and transmission by Ae. aegypti . Prior DENV and ZIKV immunity consistently suppressed YFV viremia in experimentally infected macaques, leading to reductions in Ae. aegypti infection when mosquitoes were fed on infected animals. These results indicate that, in DENV- and ZIKV-endemic regions such as South America and Asia, flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks. One-Sentence Summary: Immunity from dengue and Zika viruses suppresses yellow fever viremia, preventing infection of mosquitoes and reducing the risk of epidemics.

Zoonotic mosquito-borne arboviruses: Spillover, spillback, and realistic mitigation strategies.

Emerging zoonotic mosquito-borne viruses pose increasing health threats because of growing mosquito population, geographic expansions, and control challenges. We emphasize the need for global preparedness to effectively mitigate the health, societal, and economic impacts of spillover by these viruses through proactive measures of prediction, surveillance, prevention, and treatment.

Testicular pathological alterations associated with SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiologic agent of the coronavirus disease 2019 (COVID-19), which caused one of the pandemics with the highest mortalities with millions of deaths and hundreds of millions of cases to date. Due to its potential for airborne transmission, many studies have focused on SARS-CoV-2 primarily as a respiratory disease. However, the spread of SARS-CoV-2 to non-respiratory organs has been experimentally demonstrated and clinically observed. During autopsy studies, histopathological lesions, and disruption of the blood-testes barrier (BTB) have been observed in the male reproductive tract. Here, we review findings from both autopsy cases and animal models that demonstrate testicular disease due to COVID-19 and present an overview of the pathological alterations that occur in the testes resulting from SARS-CoV-2 infection and explore its potential mechanisms.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

Aedes aegypti Shows Increased Susceptibility to Zika Virus via Both In Vitro and In Vivo Models of Type II Diabetes.

Chronic conditions like type II diabetes (T2DM) have long been known to exacerbate many infectious diseases. For many arboviruses, including Zika virus (ZIKV), severe outcomes, morbidity and mortality usually only occur in patients with such pre-existing conditions. However, the effects of T2DM and other pre-existing conditions on human blood (e.g., hypo/hyperinsulinemia, hyperglycemia and hyperlipidemia) that may impact infectivity of arboviruses for vectors is largely unexplored. We investigated whether the susceptibility of Aedes aegypti mosquitoes was affected when the mosquitoes fed on "diabetic" bloodmeals, such as bloodmeals composed of artificially glycosylated erythrocytes or those from viremic, diabetic mice (LEPRDB/DB). Increasing glycosylation of erythrocytes from hemoglobin A1c (HgbA1c) values of 5.5-5.9 to 6.2 increased the infection rate of a Galveston, Texas strain of Ae. aegypti to ZIKV strain PRVABC59 at a bloodmeal titer of 4.14 log10 FFU/mL from 0.0 to 40.9 and 42.9%, respectively. ZIKV was present in the blood of viremic LEPRDB/DB mice at similar levels as isogenic control C57BL/6J mice (3.3 log10 FFU/mL and 3.6 log10 FFU/mL, respectively. When mice sustained a higher ZIKV viremia of 4.6 log10 FFU/mL, LEPRDB/DB mice infected 36.3% of mosquitoes while control C57BL/6J mice with a viremia of 4.2 log10 FFU/mL infected only 4.1%. Additionally, when highly susceptible Ae. aegypti Rockefeller mosquitoes fed on homozygous LEPRDB/DB, heterozygous LEPRWT/DB, and control C57BL/6J mice with viremias of ≈ 4 log10 FFU/mL, 54%, 15%, and 33% were infected, respectively. In total, these data suggest that the prevalence of T2DM in a population may have a significant impact on ZIKV transmission and indicates the need for further investigation of the impacts of pre-existing metabolic conditions on arbovirus transmission.

Venezuelan Equine Encephalitis Virus V3526 Vaccine RNA-Dependent RNA Polymerase Mutants Increase Vaccine Safety Through Restricted Tissue Tropism in a Murine Model.

Background: Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas. There are no approved vaccines or antivirals. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, albeit less so for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that altered the error incorporation rate of the RNA-dependent RNA polymerase (RdRp). Methods: The research presented here examines the impact of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains. Results: Our results show that the V3526 RdRp mutants exhibited reduced tissue tropism in the spleen and kidney compared to wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing showed that the RdRp mutations could revert to wild-type V3526. Conclusions: The observed genotypic reversion is likely of limited concern because wild-type V3526 is still an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.

Vaccination Decreases the Infectious Viral Load of Delta Variant SARS-CoV-2 in Asymptomatic Patients.

The Delta variant of SARS-CoV-2 has caused many breakthrough infections in fully vaccinated individuals. While vaccine status did not generally impact the number of viral RNA genome copies in nasopharyngeal swabs of breakthrough patients, as measured by Ct values, it has been previously found to decrease the infectious viral load in symptomatic patients. We quantified the viral RNA, infectious virus, and anti-spike IgA in nasopharyngeal swabs collected from individuals asymptomatically infected with the Delta variant of SARS-CoV-2. Vaccination decreased the infectious viral load, but not the amount of viral RNA. Furthermore, vaccinees with asymptomatic infections had significantly higher levels of anti-spike IgA in their nasal secretions compared to unvaccinated individuals with asymptomatic infections. Thus, vaccination may decrease the transmission risk of Delta, and perhaps other variants, despite not affecting the amount of viral RNA measured in nasopharyngeal swabs.

Assessing the variability of Brazilian Vaccinia virus isolates from a horse exanthematic lesion: coinfection with distinct viruses.

During the last bovine vaccinia (BV) outbreaks, several Vaccinia virus (VACV) strains were isolated and characterised, revealing significant polymorphisms between strains, even within conserved genes. Although the epidemiology of VACV has been studied in BV outbreaks, there is little data about the circulation of the Brazilian VACV isolates. This study describes the genetic and biological characterisation of two VACV isolates, Pelotas 1 virus (P1V) and Pelotas 2 virus (P2V), which were obtained concomitantly from a horse affected by severe cutaneous disease. Despite being isolated from the same exanthematic clinical sample, P1V and P2V showed differences in their plaque phenotype and in one-step growth curves. Moreover, P1V and P2V presented distinct virulence profiles in a BALB/c mouse model, as observed with other Brazilian VACV isolates. Sequencing and phylogenetic analysis of four different genes demonstrated that the isolates are segregated in different VACV clusters. Our results raise interesting questions about the diversity of VACV isolates in Brazil.

SARS-CoV-2 Infects Hamster Testes.

The COVID-19 pandemic continues to affect millions of people worldwide. Although SARS-CoV-2 is a respiratory virus, there is growing concern that the disease could cause damage and pathology outside the lungs, including in the genital tract. Studies suggest that SARS-CoV-2 infection can damage the testes and reduce testosterone levels, but the underlying mechanisms are unknown and evidence of virus replication in testicular cells is lacking. We infected golden Syrian hamsters intranasally, a model for mild human COVID-19, and detected viral RNA in testes samples without histopathological changes up to one month post-infection. Using an ex vivo infection model, we detected SARS-CoV-2 replication in hamster testicular cells. Taken together, our data raise the possibility that testes damage observed in severe cases of COVID-19 could be partly explained by direct SARS-CoV-2 infection of the testicular cells.

Vaccinia virus infection in monkeys, Brazilian Amazon.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

Vaccinia virus is not inactivated after thermal treatment and cheese production using experimentally contaminated milk.

Bovine vaccinia is an emergent zoonosis caused by the Vaccinia virus (VACV). The disease is characterized by the appearance of exanthematic lesions that occur in humans and dairy cows. Previous studies have revealed the presence of infectious viral particles in milk samples during an outbreak of bovine vaccinia in Brazil, indicating the possibility of disease transmission through raw milk. To assess the viability of the virus in milk after thermal treatment and processing procedures, milk samples were experimentally contaminated with 10(3) plaque forming units (PFU)/mL (group I) and 10(5) PFU/mL (group II) VACV Guarani P2 virus, and the third group was not contaminated and served as a control. The samples were submitted to storage temperatures in a cold chamber, freezer for 48 hours, and to low temperature long-time treatment. Moreover, the viral viability was evaluated in cheese produced with contaminated milk using 10(4) PFU/mL VACV Guarani P2. Notably, the virus remained viable in milk after storage for 48 hours in both the cold chamber and the freezer, with a reduction in viral titer of 14.49% and 25.86%, respectively. Group II showed a viral reduction in titer of 61.88% and 75.98%, respectively. Thermal treatment 65°C for 30 minutes showed a reduction of viral titer of 94.83% and 99.99%, respectively, in group I and group II, but still showed remaining viable virus particles. In addition, it was possible to recover infectious viral particles from both the solid curds and the whey of the cheese produced with experimentally contaminated milk. The cheese shows a reduction in viral titer of 84.87% after storage at 4°C for 24 hours. The presence of viable viral particles in milk after both thermal treatment and cheese production indicates a potential public health risk.

Epidemic Alphaviruses: Ecology, Emergence and Outbreaks.

Over the past century, the emergence/reemergence of arthropod-borne zoonotic agents has been a growing public health concern. In particular, agents from the genus Alphavirus pose a significant risk to both animal and human health. Human alphaviral disease presents with either arthritogenic or encephalitic manifestations and is associated with significant morbidity and/or mortality. Unfortunately, there are presently no vaccines or antiviral measures approved for human use. The present review examines the ecology, epidemiology, disease, past outbreaks, and potential to cause contemporary outbreaks for several alphavirus pathogens.

Decontamination of SARS-CoV-2 and Other RNA Viruses from N95 Level Meltblown Polypropylene Fabric Using Heat under Different Humidities.

In March of 2020, the World Health Organization declared a pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The pandemic led to a shortage of N95-grade filtering facepiece respirators (FFRs), especially surgical-grade N95 FFRs for protection of healthcare professionals against airborne transmission of SARS-CoV-2. We and others have previously reported promising decontamination methods that may be applied to the recycling and reuse of FFRs. In this study we tested disinfection of three viruses, including SARS-CoV-2, dried on a piece of meltblown fabric, the principal component responsible for filtering of fine particles in N95-level FFRs, under a range of temperatures (60-95 °C) at ambient or 100% relative humidity (RH) in conjunction with filtration efficiency testing. We found that heat treatments of 75 °C for 30 min or 85 °C for 20 min at 100% RH resulted in efficient decontamination from the fabric of SARS-CoV-2, human coronavirus NL63 (HCoV-NL63), and another enveloped RNA virus, chikungunya virus vaccine strain 181/25 (CHIKV-181/25), without lowering the meltblown fabric's filtration efficiency.