High-level Replication Research Articles

SARS-CoV-2 evolution balances conflicting roles of N protein phosphorylation.

All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection.

The Core of the Matter-Importance of Identification Method and Biological Replication for Benthic Marine Monitoring.

Benthic macrofauna are important and widely used biological indicators of marine ecosystems as they have limited mobility and therefore integrate the effects of local environmental stressors over time. Recently, environmental DNA (eDNA) analysis has provided a potentially more resource-efficient approach for benthic biomonitoring than traditional morphology-based methods. Several studies have compared eDNA with morphology-based monitoring, but few have compared the two approaches using the exact same sediment cores. In addition, the meiofauna and pelagic organisms obtained as 'bycatch' using eDNA have largely been disregarded from comparisons. Here, we address these shortcomings through comparative invertebrate analyses of six sediment sample replicates from each of four stations in Denmark, using eDNA metabarcoding and morphological identification. Our results revealed large variation between the six replicates for both methods and little overlap in taxon compositions between methods. While the morphological dataset was dominated by molluscs and annelids, the eDNA dataset was dominated by arthropods and annelids. Using community composition data, we found that sampling stations could be distinguished both with eDNA and morphology. Finally, we inferred expected total richness from extrapolated accumulation curves of detected taxa from each method. This indicated that eDNA metabarcoding requires less replication than morphology for maximum coverage of diversity to be reached. However, both methods required high levels of replication, and our results on taxonomic composition add to the evidence that morphological and eDNA-based methods should preferably be used as complimentary tools for marine bioassessment.

The Human Neural Cell Atlas of Zika Infection in developing human brain tissue: viral pathogenesis, innate immunity, and lineage reprogramming.

Zika virus (ZIKV) infection during pregnancy can lead to fetal brain infection and developmental anomalies collectively known as congenital Zika syndrome (CZS). To define the molecular features underlying CZS in a relevant human cell model, we evaluated ZIKV infection and neurodevelopment in primary fetal brain explants and induced pluripotent stem cell-derived mixed neural cultures at single cell resolution. We identified astrocytes as key innate immune sentinel cells detecting ZIKV and producing IFN-β. In contrast, neural progenitor cells displayed impaired innate immunity and supported high levels of viral replication. ZIKV infection of neurons suppressed differentiation and synaptic signaling networks and programmed a molecular switch from neurogenesis to astrogliogenesis. We identified a universal ZIKV-driven cellular stress response linked to intrinsic apoptosis and regulated by IFN-β. These findings reveal how innate immune signaling intersects with ZIKV-driven perturbations in cellular function to influence CZS outcomes including neuron developmental dysfunction and apoptotic cell death.

Continual Day–Night eDNA Detectability Amidst Diel Reef Species Fluctuations on Diver Transects

ABSTRACTRecent research into the spatiotemporal dynamics of eDNA in marine environments indicates that eDNA signals are highly localized and may dissipate beyond detection levels within a few hours of production. This affects whether single‐timepoint eDNA sampling, which generally occurs during daylight hours, or cyclic (day/night) interval eDNA sampling is necessary to detect both diurnal and nocturnal marine species. Our study investigated short‐term variability in eDNA derived from fishes and macroinvertebrates across three temperate reef sites in eastern Tasmania, Australia. Simultaneous eDNA and underwater visual census (UVC) diver transect surveys were conducted every 6 h over a 24‐h period to investigate whether eDNA was able to detect marine species outside of their UVC‐observed diel activity. We report that single‐timepoint eDNA sampling can detect both diurnal and nocturnal species on temperate reefscapes. A lack of eDNA compositional turnover between day and night suggests that eDNA persists beyond 12 h and/or is continuously produced by both diurnal and nocturnal reef taxa, irrespective of diel behavioral changes observed by UVC. Given high eDNA sample variability, however, we recommend a high replication level (> 10 × 1 L samples) to produce robust site community composition profiles. This study builds on emerging literature on short‐term variability in eDNA, assisting in the design of future eDNA studies at sites with pronounced variation in faunal activity between day and night.

Immunomodulatory soluble HLA-G and HLA-E are associated with rapidly deteriorating CLAD and HCMV viremia after lung transplantation

Plasma-soluble (s)HLA-G and sHLA-E are immunoregulatory proteins that balance the activation of NKG2A+ immune cells. In lung-transplant recipients (LTRs), dysregulated NKG2A+ natural killer cell responses may result in high-level HCMV replication as well as chronic lung allograft dysfunction (CLAD), and especially the development of rapidly deteriorating CLAD is associated with high mortality. We thus analyzed the kinetics and function of sHLA-G and sHLA-E in follow-up samples of N=76 LTRs to evaluate whether these immunoregulatory proteins are associated with the risk for CLAD and high-level HCMV replication. Here, we demonstrate that rapidly deteriorating CLAD LTRs are hallmarked by continually low (<107 ng/mL) sHLA-G levels. In contrast, high sHLA-E levels were associated with the following development of high-level (>1000 copies/mL) HCMV episodes. Thus, sHLA-G and sHLA-E may serve as novel biomarkers for the development of rapidly deteriorating CLAD and high-level HCMV replication in LTRs.

HOXA-AS2 Epigenetically Inhibits HBV Transcription by Recruiting the MTA1-HDAC1/2 Deacetylase Complex to cccDNA Minichromosome.

Persistent transcription of HBV covalently closed circular DNA (cccDNA) is critical for chronic HBV infection. Silencing cccDNA transcription through epigenetic mechanisms offers an effective strategy to control HBV. Long non-coding RNAs (lncRNAs), as important epigenetic regulators, have an unclear role in cccDNA transcription regulation. In this study, lncRNA sequencing (lncRNA seq) is conducted on five pairs of HBV-positive and HBV-negative liver tissue. Through analysis, HOXA-AS2 (HOXA cluster antisense RNA 2) is identified as a significantly upregulated lncRNA in HBV-infected livers. Further experiments demonstrate that HBV DNA polymerase (DNA pol) induces HOXA-AS2 after establishing persistent high-level HBV replication. Functional studies reveal that HOXA-AS2 physically binds to cccDNA and significantly inhibits its transcription. Mechanistically, HOXA-AS2 recruits the MTA1-HDAC1/2 deacetylase complex to cccDNA minichromosome by physically interacting with metastasis associated 1 (MTA1) subunit, resulting in reduced acetylation of histone H3 at lysine 9 (H3K9ac) and lysine 27 (H3K27ac) associated with cccDNA and subsequently suppressing cccDNA transcription. Altogether, the study reveals a mechanism to self-limit HBV replication, wherein the upregulation of lncRNA HOXA-AS2, induced by HBV DNA pol, can epigenetically suppress cccDNA transcription.

A Field Survey of Grapevine Germplasm Susceptible to Eutypa Dieback

Eutypa dieback of grapevine is a trunk disease that impacts vineyard productivity worldwide. Grape germplasm is typically evaluated for resistance to Eutypa dieback through controlled inoculations in the greenhouse, although the high level of replication required of this approach (40 plants per genotype) can limit the total number of genotypes evaluated. An alternative approach is to evaluate naturally infected genotypes in the field. We rated the incidence and severity of vines with the diagnostic leaf symptoms of Eutypa dieback and the incidence of mortality among such vines of 973 Vitis vinifera accessions (planted in duplicate) at the US Department of Agriculture, National Clonal Germplasm Repository in Davis, CA, USA, which is maintained as a living collection for grape research. Across 3 years and spanning a total of 5 years (2011, 2013, and 2015), 120 accessions had leaf symptoms in one or more years (“susceptible accessions”). Courbu blanc [Davis Vitis identification tag (DVIT) 2313], Frankenthal blanc (DVIT 2115), and Pinot gris (DVIT 0907) were the only accessions with leaf symptoms each year. Accessions with the most severe leaf symptoms (a rating of 5 points) were Chasselas Napoleon (DVIT 0375) and Queen of the Vineyard (DVIT 0496). We identified susceptible accessions—namely, those related to ‘Chasselas’ and ‘Muscat’—with a shared genetic background, based on a previous single nucleotide polymorphism genotyping effort of the collection. Especially for grapevine, a long-lived perennial that is meant to produce a crop for decades, knowledge of susceptible accessions and their pedigrees can help inform breeding programs and studies on the host response to infection.

TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n=16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2=0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

The Digestive Vacuole of the Malaria Parasite: A Specialized Lysosome.

The malaria parasite resides within erythrocytes during one stage of its life cycle. During this intraerythrocytic period, the parasite ingests the erythrocyte cytoplasm and digests approximately two-thirds of the host cell hemoglobin. This digestion occurs within a lysosome-like organelle called the digestive vacuole. Several proteases are localized to the digestive vacuole and these proteases sequentially breakdown hemoglobin into small peptides, dipeptides, and amino acids. The peptides are exported into the host cytoplasm via the chloroquine-resistance transporter and an amino acid transporter has also been identified on the digestive vacuole membrane. The environment of the digestive vacuole also provides appropriate conditions for the biocrystallization of toxic heme into non-toxic hemozoin by a poorly understood process. Hemozoin formation is an attribute of Plasmodium and Haemoproteus and is not exhibited by other intraerythrocytic protozoan parasites. The efficient degradation of hemoglobin and detoxification of heme likely plays a major role in the high level of replication exhibited by malaria parasites within erythrocytes. Unique features of the digestive vacuole and the critical importance of nutrient acquisition provide therapeutic targets for the treatment of malaria.

Straight to the point: evaluation of a Point of Care Information (POCI) resource in answering disease-related questions.

To evaluate the ability of DynaMedex, an evidence-based drug and disease Point of Care Information (POCI) resource, in answering clinical queries using keyword searches. Real-world disease-related questions compiled from clinicians at an academic medical center, DynaMedex search query data, and medical board review resources were categorized into five clinical categories (complications & prognosis, diagnosis & clinical presentation, epidemiology, prevention & screening/monitoring, and treatment) and six specialties (cardiology, endocrinology, hematology-oncology, infectious disease, internal medicine, and neurology). A total of 265 disease-related questions were evaluated by pharmacist reviewers based on if an answer was found (yes, no), whether the answer was relevant (yes, no), difficulty in finding the answer (easy, not easy), cited best evidence available (yes, no), clinical practice guidelines included (yes, no), and level of detail provided (detailed, limited details). An answer was found for 259/265 questions (98%). Both reviewers found an answer for 241 questions (91%), neither found the answer for 6 questions (2%), and only one reviewer found an answer for 18 questions (7%). Both reviewers found a relevant answer 97% of the time when an answer was found. Of all relevant answers found, 68% were easy to find, 97% cited best quality of evidence available, 72% included clinical guidelines, and 95% were detailed. Recommendations for areas of resource improvement were identified. The resource enabled reviewers to answer most questions easily with the best quality of evidence available, providing detailed answers and clinical guidelines, with a high level of replication of results across users.

Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of thehepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

Adipose Tissues from Human and Bat-Derived Cell Lines Support Ebola Virus Infection.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat (Eptesicus fuscus) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

T cell activation is insufficient to drive SIV disease progression.

Resolution of T cell activation and inflammation is a key determinant of the lack of SIV disease progression in African green monkeys (AGMs). Although frequently considered together, T cell activation occurs in response to viral stimulation of acquired immunity, while inflammation reflects innate immune responses to mucosal injury. We dissociated T cell activation from inflammation through regulatory T cell (Treg) depletion with Ontak (interleukin-2 coupled with diphtheria toxin) during early SIV infection of AGMs. This intervention abolished control of T cell immune activation beyond the transition from acute to chronic infection. Ontak had no effect on gut barrier integrity, microbial translocation, inflammation, and hypercoagulation, despite increasing T cell activation. Ontak administration increased macrophage counts yet decreased their activation. Persistent T cell activation influenced SIV pathogenesis, shifting the ramp-up in viral replication to earlier time points, prolonging the high levels of replication, and delaying CD4+ T cell restoration yet without any clinical or biological sign of disease progression in Treg-depleted AGMs. Thus, by inducing T cell activation without damaging mucosal barrier integrity, we showed that systemic T cell activation per se is not sufficient to drive disease progression, which suggests that control of systemic inflammation (likely through maintenance of gut integrity) is the key determinant of lack of disease progression in natural hosts of SIVs.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export.

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

Use of Zebrafish Embryos To Reproduce Human Norovirus and To Evaluate Human Norovirus Infectivity Decay after UV Treatment.

This study reports an essential improvement of the method for replication of human norovirus (HNoV) with the use of zebrafish (Danio rerio) embryos. With three HNoV genotypes and P-types GII.2[P16], GII.4[P16], and GII.17[P31], we demonstrated that this tool had higher efficiency and robustness than the zebrafish larvae as reported previously. When zebrafish larvae were injected with virus (1.6 ± 0.3 log genome copies/10 larvae), a significant increase of virus genome copies was detected at 2 days postinfection (dpi; 4.4 ± 0.8 log genome copies/10 larvae, P < 0.05) and the viral loads started to decrease gradually from 3 dpi. In comparison, when the viruses were injected into the zebrafish embryos, significant virus replication was noticed from 1 dpi and lasted to 6 dpi (P < 0.05). The virus levels detected at 3 dpi had the highest mean value and the smallest variation (7.7 ± 0.2 log genome copies/10 larvae). The high levels of virus replication enabled continuous passaging for all three strains up to four passages. The zebrafish embryo-generated HNoVs showed clear patterns of binding to human histo-blood group antigens (HBGAs) in human saliva by a simple saliva-binding reverse transcription-quantitative PCR (RT-qPCR). Last, in a disinfection study, it was shown that a dose of 6 mJ/cm2 UV254 was able induce a >2-log reduction in HNoV infectivity for all three HNoV strains tested, suggesting that HNoVs were more UV susceptible than multiple enteric viruses and commonly used HNoV surrogates as tested before. IMPORTANCE HNoVs are a leading cause of gastroenteritis outbreaks worldwide. The zebrafish embryo tool as developed in this study serves as an efficient way to generate viruses with high titers and clean background and a straightforward platform to evaluate HNoV inactivation efficacies. It is expected that this tool will not only benefit epidemiological research on HNoV but also be used to generate HNoV inactivation parameters which are highly needed by the water treatment and food industries.

Regulation of Virus Replication by BK Polyomavirus Small T Antigen.

Polyomavirus small T antigen (tAg) plays important roles in regulating viral replication, the innate immune response, apoptosis, and transformation for SV40, Merkel cell polyomavirus (MCPyV), murine polyomavirus (MuPyV), and JC polyomavirus (JCPyV). However, the function of BK polyomavirus (BKPyV) tAg has been much less studied. Here, we constructed mutant viruses that do not express tAg, and we showed that, in contrast with other polyomaviruses, BKPyV tAg inhibits large T antigen (TAg) gene expression and viral DNA replication. However, this occurs only in an archetype viral background. We also observed that the transduction of cells with a lentivirus-expressing BKPyV tAg kills the cells. We further discovered that BKPyV tAg interacts not only with PP2A A and C subunits, as has been demonstrated for other polyomavirus tAg proteins, but also with PP2A B''' subunit members. Knocking down either of two B''' subunits, namely STRN or STRN3, mimics the phenotype of the tAg mutant virus. However, a virus containing a point mutation in the PP2A binding domain of tAg only partially affected virus TAg expression and DNA replication. These results indicate that BKPyV tAg downregulates viral gene expression and DNA replication and that this occurs in part through interactions with PP2A. IMPORTANCE BK polyomavirus is a virus that establishes a lifelong infection of the majority of people. The infection usually does not cause any clinical symptoms, but, in transplant recipients whose immune systems have been suppressed, unchecked virus replication can cause severe disease. In this study, we show that a viral protein called small T antigen is one of the ways that the virus can persist without high levels of replication. Understanding which factors control viral replication enhances our knowledge of the virus life cycle and could lead to potential interventions for these patients.