Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

Stability of Enveloped and Nonenveloped Viruses in a Stable Gelatin Liquid Formulation

The thermal stability of relevant viruses in gelatin liquid formulations for medical research and application is poorly understood. Bovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus stable liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus (PIV), reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period. The BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV (log10 reduction value) at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin (60000 and 160000) with an LRV above 1. Based on the gelatin type, BHV in B-type gelatin samples were unexpectantly more stable than in A-type gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks. The results demonstrated that 5% hydrolyzed gelatin can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.

Recognition of HIV-1 capsid licenses innate immune response to viral infection

Cyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1-encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.

Current Understanding of the Pathogenesis of Porcine Circovirus 3

Circoviruses are closed, circular, single-stranded DNA viruses belonging to the family Circoviridae and the genus Circovirus. To date, at least four porcine circoviruses (PCVs) have been recognized, including PCV1 to PCV4, respectively. Similar to PCV2 pathogenesis, PCV3 has been reported worldwide with myriad clinical and pathological presentations such as reproductive disorders, respiratory diseases, diarrhea etc. Current understanding of PCV3 pathogenesis is very limited since the majority of studies were mostly field observations. Interpretation of the results from such studies is not always simple. Various confounding factors affect the clinical appearance and pathological changes of the infected pigs. Recently, several experimental PCV3 infection studies have been reported, providing a better understanding of its pathogenesis. In this review, we focused on novel findings regarding PCV3 pathogenesis from both field observation and experimental infection studies. Possible factors involved in the conflicting results among the experimental infection studies are also discussed. This review article provides important insight into the current knowledge on PCV3 pathogenesis which would aid in prioritizing research in order to fill the knowledge gaps.

The microRNA days: The COVID-19 pandemic from the point of view of short RNAs

Despite their biological simplicity, microRNA-based organisms, such as RNA viruses, are currently shown to be unexpected threats to mammals, including humans. This situation is exemplified by the COVID-19 pandemic triggered by the spread of SARS-CoV-2. RNA viruses are older than DNA viruses. Indeed, from an evolutionary standpoint, RNA is an older molecule than DNA. The strength of RNA viruses, compared to DNA viruses, resides in their simplicity and instability. The instability of RNA viruses, such as human immunodeficiency virus (HIV) and flu viruses, generates mutants to escape the host’s defense mechanisms. A formidable combination of lethality and infectivity was recently achieved by SARS-CoV-2. Complex DNAbased defense systems use Toll-like receptors to intercept viral RNA inside a cell. Activation of Toll-like receptors triggers inflammation and activates lymphocytes and monocytes, causing thromboxane release. In the case of SARS-CoV-2 infection, this process results in cytokine storms and lung thromboembolism. The ongoing pandemic can be envisioned as a struggle between highly evolved complex DNA organisms, i.e., humans, and poorly evolved simple RNA organisms, i.e., SARS-CoV-2 virus. Quite surprisingly, the complex organism has a serious problem defeating the simplistic organism. However, humans are finally developing a new effective weapon in fighting the SARS-CoV-2 virus, paradoxically, RNA-based vaccines. These considerations underscore the relevance of microRNAs as powerful tools in therapeutic and preventive medicine.

Discovery of a class of giant virus relatives displaying unusual functional traits and prevalent within plankton: the Mirusviricetes

Large and giant DNA viruses of the phylum Nucleocytoviricota have a profound influence on the ecology and evolution of planktonic eukaryotes. Recently, various Nucleocytoviricota genomes have been characterized from environmental metagenomes based on the occurrence of hallmark genes identified from cultures. However, lineages diverging from the culture genomics functional principles have been overlooked thus far. Here, we developed a phylogeny-guided genome-resolved metagenomic framework using a single hallmark gene as compass, a subunit of DNA-dependent RNA polymerase encoded by most Nucleocytoviricota. We applied this method to large metagenomic data sets from the surface of five oceans and two seas and characterized 697 non-redundant Nucleocytoviricota genomes up to 1.45 Mbp in length. This database expands the known diversity of the class Megaviricetes and revealed two additional putative classes we named Proculviricetes and Mirusviricetes. Critically, the diverse and prevalent Mirusviricetes population genomes seemingly lack several hallmark genes, in particular those related to viral particle morphogenesis. Instead, they share various genes of known (e.g., TATA-binding proteins, histones, proteases and viral rhodopsins) and unknown functions rarely detected if not entirely missing in all other characterized Nucleocytoviricota lineages. Phylogenomics, comparative genomics, functional trends and the signal among planktonic cellular size fractions point to Mirusviricetes being a major, functionally divergent class of large DNA viruses that actively infect eukaryotes in the sunlit ocean using an enigmatic functional life style. Finally, we built a comprehensive marine genomic database for Nucleocytoviricota by combining multiple environmental surveys that might contribute to future endeavors exploring the ecology and evolution of plankton.

A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

First record of ranavirus (Ranavirus sp.) in Siberia, Russia

Ranaviruses are a group of double-strand DNA viruses that infect fish, amphibians and reptiles. These viruses are responsible for mass fish and amphibian mortality events worldwide, both in the wild and at the fish and amphibian farms. The number of detected epizootics has grown significantly in recent years. In Eastern Europe and Northern Asia, including Russia, very few ranavirosis monitoring studies have been conducted, in contrast with Western Europe and America. In the present work, we used a qPCR assay to survey for the first time the amphibian populations of West Siberia (Russia) for the presence of ranaviruses. In total, we studied 252 tissue samples from six amphibian species, collected across West Siberia from the south to the Arctic regions. We report a single infected sample: a common toad (Bufo bufo) captured near Tyumen city. The phylogenetic analysis showed that the detected virus strain belongs to the CMTV lineage. This is only the second observation of Ranavirus in Russia.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named ‘Suismacovirus’, comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses’ genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.