The feasibility of field collected pig oronasal secretions as specimens for the virologic surveillance of Japanese encephalitis virus

Virologic surveillance of Japanese encephalitis virus (JEV) relies on collecting pig blood specimens and adult mosquitoes in the past. Viral RNAs extracted from pig blood specimens suffer from low detecting positivity by reverse transcription PCR (RT-PCR). The oronasal transmission of the virus has been demonstrated in experimentally infected pigs. This observation suggested oronasal specimens could be useful source in the virus surveillance. However, the role of this unusual route of transmission remains unproven in the operational pig farm. In this study, we explore the feasibility of using pig oronasal secretions collected by chewing ropes to improve the positivity of detection in commercial pig farms. The multiplex genotype-specific RT-PCR was used in this study to determine and compare the positivity of detecting JEV viral RNAs in pig’s oronasal secretions and blood specimens, and the primary mosquito vector. Oronasal specimens had the overall positive rate of 6.0% (95% CI 1.3%–16.6%) (3/50) to 10.0% (95% CI 2.1%–26.5%) (3/30) for JEV during transmission period despite the negative results of all blood-derived specimens (n = 2442). Interestingly, pig oronasal secretions and female Culex tritaeniorhynchus mosquito samples collected from the same pig farm showed similar viral RNA positive rates, 10.0% (95% CI 2.1%–26.5%) (3/30) and 8.9% (95% CI 2.5%–21.2%) (4/45), respectively (p> 0.05). Pig oronasal secretion-based surveillance revealed the seasonality of viral activity and identified closely related genotype I virus derived from the mosquito isolates. This finding indicates oronasal secretion-based RT-PCR assay can be a non-invasive, alternative method of implementing JEV surveillance in the epidemic area prior to the circulation of virus-positive mosquitoes.

NUDT2 initiates viral RNA degradation by removal of 5′-phosphates

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.

Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond

RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture

Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.

The antiviral state has shaped the CpG composition of the vertebrate interferome to avoid self-targeting

Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

Structural analysis of 3′UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3′UTRs and their ability to produce subgenomic flaviviral RNAs — noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5′-3′ exoribonuclease XRN1. We determined the secondary structures of complete 3′UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3′UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

De-Coding the Contributions of the Viral RNAs to Alphaviral Pathogenesis

Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease.

RNA-Binding Proteins at the Host-Pathogen Interface Targeting Viral Regulatory Elements

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.