Viral Genome Copy Number Research Articles

Research on expression patterns of endogenous OASL and IFN-α in duck embryos infected with DHAV-3.

An animal body's immune response to viruses might vary depending on various factors. The relationship between 2'-5'-oligoadenylate synthetase like (OASL), interferon alpha (IFN-α), and duck hepatitis A virus type 3 (DHAV-3) virus genome copy number in duck embryo tissues was studied to investigate duck's natural antiviral immunity mechanism. 15-day-old SPF duck embryos were infected with DHAV-3 and their organs and tissues were collected at various times after inoculation. RT real-time PCR was used to determine OASL and IFNA mRNA expression levels and the DHAV-3 copy number. Compared with that at 0 hours, liver OASL transcription increased significantly at 24 hours, and extremely significantly in the liver, heart, gizzard, small intestine, and muscle at 48 hours. The heart had the highest expression level, followed by the liver, with lower expression in the other tissues. There was no significant difference in IFNA expression between the heart and liver at 12 hours, but it started to rise at 24 hours, reaching its maximum at 48 hours. Although IFNA expression increased in the gizzards, small intestines, and muscles, its relative expression levels were lower than those in the heart and liver. In the tissues, the virus genome copy number peaked at 24 hours, and then decreased. The liver had the highest virus genome copy number, followed by the heart. The results implied that the differences in OASL transcription in the tested tissues were similar to those of IFNA. Thus, the immune response (OASL and IFN-α levels) of duck embryos to DHAV-3 shows tissue differences, especially in the liver and heart.

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Evaluation of rapid amplicon-based nanopore sequencing using the latest chemistry for accurate whole genome analysis of influenza A virus in clinical samples

MinION sequencing is widely used to sequence influenza A virus (IAV) genomes; however, the accuracy and utility of this approach, using the latest chemistry to obtain whole viral genome sequences directly from clinical samples, remain insufficiently investigated. We evaluated the sequencing accuracy of combining simultaneous multisegment one-step RT-PCR and MinION sequencing using various subtypes of 13 IAV isolates. The latest R10.4.1 chemistry significantly improved sequencing accuracy, achieving ≥99.993% identity with Illumina MiSeq results and reducing the single nucleotide deletion in homopolymer regions. Applying this method to 11 clinical samples enabled rapid subtype identification and the acquisition of eight full-length IAV genomes. In four of these samples, subtype identification of HA and NA was achieved within 20 min after the start of sequencing and a full-length IAV genome was obtained within 7 h after RNA extraction. However, there was concern that cross barcode misassignment during demultiplexing affected data interpretation, particularly for samples with low viral genome copy numbers. This approach can be used for the rapid identification of IAV subtypes and accurate acquisition of full IAV genome sequences from clinical samples, although careful data analysis is required for the multiplex sequencing of clinical samples with low viral genome copy numbers.

The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response.

The roles of host cell miRNAs have not been well studied in the context of BCoV replication and immune regulation. This study aimed to identify miRNA candidates that regulate essential host genes involved in BCoV replication, tissue tropism, and immune regulation. To achieve these goals, we used two isolates of BCoV (enteric and respiratory) to infect bovine endothelial cells (BECs) and Madine Darby Bovine Kidney (MDBK) cells. We determined the miRNA expression profiles of these cells after BCoV infection. The expression of miRNA16a is differentially altered during BCoV infection. Our data show that miRNA16a is a significantly downregulated miRNA in both in vitro and ex vivo models. We confirmed the miRNA16aexpression profile by qRT-PCR. Overexpression of pre-miRNA16ain the BEC and the MDBK cell lines markedly inhibited BCoV infection, as determined by the viral genome copy numbers measured by qRT‒PCR, viral protein expression (S and N) measured by Western blot, and virus infectivity using a plaque assay. Our bioinformatic prediction showed that Furin is a potential target of miRNA16a. We compared the Furin protein expression level in pre-miRNA16a-transfected/BCoV-infected cells to that in pre-miRNA-scrambled-transfected cells. Our qRT-PCR and Western blot data revealed marked inhibition of Furin expression at the mRNA and protein levels, respectively. BCoV-S protein expression was markedly inhibited at both the mRNA and protein levels. To further confirm the impact of the downregulation of the Furin enzyme on the replication of BCoV, we transfected cells with specific Furin-siRNAs parallel to the scrambled siRNA. Marked inhibition of BCoV replication was observed in the Furin-siRNA-treated group. To further validate Furin as a novel target for miRNA16a, we cloned the 3'UTR of bovine Furin carrying the seed region of miRNA16a in the dual luciferase vector. Our data showed that luciferase activity in pre-miRNA16a-transfected cells decreased by more than 50% compared to cells transfected with the construct carrying the mutated Furin seed region. Our data confirmed that miRNA16ainhibits BCoV replication by targeting the host cell line Furin and the BCoV-S glycoprotein. It also enhances the host immune response, which contributes to the inhibition of viral replication. This is the first study to confirm that Furin is a valid target of miRNA16a. Our findings highlight the clinical applications of host miRNA16a as a potential miRNA-based vaccine/antiviral therapy.

Efficient Simulation of Viral Transduction and Propagation for Biomanufacturing.

The design of biomanufacturing platforms based on viral transduction and/or propagation poses significant challenges at the intersection between synthetic biology and process engineering. This paper introduces vitraPro, a software toolkit composed of a multiscale model and an efficient numeric technique that can be leveraged for determining genetic and process designs that optimize transduction-based biomanufacturing platforms and viral amplification processes. Viral infection and propagation for up to two viruses simultaneously can be simulated through the model, considering viruses in either the lytic or lysogenic stage, during batch, perfusion, or continuous operation. The model estimates the distribution of the viral genome(s) copy number in the cell population, which is an indicator of transduction efficiency and viral genome stability. The infection age distribution of the infected cells is also calculated, indicating how many cells are in an infection stage compatible with recombinant product expression or viral amplification. The model can also consider the presence of defective interfering particles in the system, which can severely compromise the productivity of biomanufacturing processes. Model benchmarking and validation are demonstrated for case studies of the baculovirus expression vector system and influenza A propagation in suspension cultures.

Direct genome sequencing of respiratory viruses from low viral load clinical specimens using the target capture sequencing technology.

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.

Differential requirement of the transcription factor HOXC13 for the stable maintenance of human papillomavirus genome among high-risk genotypes

Differential requirement of the transcription factor HOXC13 for the stable maintenance of human papillomavirus genome among high-risk genotypes

Time-Course Changes of the Viral Genome Copy Numbers and ELISA Antibody Titer in Coho Salmon Artificially Infected with PRV-2

Time-Course Changes of the Viral Genome Copy Numbers and ELISA Antibody Titer in Coho Salmon Artificially Infected with PRV-2

Development and application of a high-sensitivity immunochromatographic test strip for detecting pseudorabies virus.

Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput. This study was designed to produce monoclonal antibodies targeting PRV through immunization of mice using the eukaryotic system to express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips. The strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336 × 103 copies/μL (the number of viral genome copies per microliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality.

Surveillance of Wastewater to Monitor the Prevalence of Gastroenteritis Viruses in Chiba Prefecture (2014–2019)

In Japan, sentinel surveillance is used to monitor the trend of infectious gastroenteritis. Another method of pathogen surveillance, wastewater-based epidemiology, has been used recently because it can help to monitor infectious disease without relying on patient data. Here, we aimed to determine the viral trends reflected in the number of reported patients and number of gastroenteritis virus-positive samples. We focused on gastroenteritis viruses present in wastewater and investigated the usefulness of wastewater surveillance for the surveillance of infectious gastroenteritis. Real-time polymerase chain reaction was used for viral gene detection in wastewater. The number of reported patients per pediatric sentinel site and number of viral genome copies were compared for correlation potential. The number of gastroenteritis virus-positive samples reported by National Epidemiological Surveillance of Infectious Disease (NESID) and the status of gastroenteritis viruses detected in wastewater were also evaluated. Genes of norovirus genotype I, norovirus genotype II, sapovirus, astrovirus, rotavirus group A, and rotavirus group C were detected in wastewater samples. Viruses were detected in wastewater during periods when no gastroenteritis virus-positive samples were reported to NESID. Norovirus genotype II and other gastroenteritis viruses were detected in wastewater even during periods when no gastroenteritis virus-positive samples were found. Therefore, surveillance using wastewater can complement sentinel surveillance and is an effective tool for the surveillance of infectious gastroenteritis.

Shrimp SIRT4 promotes white spot syndrome virus replication

Shrimp SIRT4 promotes white spot syndrome virus replication

Two Distinct Genotypes of Spissistilus festinus (Say, 1830) Reproduce and Differentially Transmit Grapevine Red Blotch Virus.

Two phenotypically similar but genetically distinct genotypes of Spissistilus festinus (Say, 1830) (Hemiptera: Membracidae), a pest of legume crops in Southern United States and a vector of grapevine red blotch virus (GRBV) in California vineyards, exist. No information is available on whether the two S. festinus genotypes, i.e., California (CA) and Southeastern (SE), are sexually compatible or whether the SE genotype can transmit GRBV. In this study, we established mixed mating S. festinus pairs for which the F1 offspring varied phenotypically compared with the offspring of same genotype pairs but acquired GRBV isolate NY175 at similar rates (p = 0.96) and with a similar viral genome copy number (p = 0.34). Likewise, rates of GRBV acquisition were alike for the two parental CA (58%, 61/105) and SE (61%, 65/106) genotypes (p = 0.74), though the GRBV copy number in the salivary glands was overall significantly higher for SE than CA individuals (p = 0.02). Furthermore, the GRBV transmission rate was significantly higher for the SE genotype (89%, 16/18) than the CA genotype (50%, 8/16) (p = 0.04). These results revealed the existence of two sexually compatible S. festinus genotypes with distinct GRBV transmission abilities, suggesting the need to study GRBV ecology in Southeastern United States and areas where the two genotypes might co-exist.

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/μg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring

Tracking community infection dynamics of COVID-19 by monitoring SARS-CoV-2 RNA in wastewater, counting positive reactions by qPCR

Tracking community infection dynamics of COVID-19 by monitoring SARS-CoV-2 RNA in wastewater, counting positive reactions by qPCR

Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19

It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID-19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.

Minimal vertical transmission of Equid alphaherpesvirus 1 (EHV-1) in normal developing foals during a naturally occurring abortion outbreak

Minimal vertical transmission of Equid alphaherpesvirus 1 (EHV-1) in normal developing foals during a naturally occurring abortion outbreak

Experimental Support for Human Papillomavirus Genome Amplification Early after Infectious Delivery.

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate-early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using 5-ethynyl-2'-deoxyuridine (EdU) pulse-labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1- and E2-dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number, confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In the absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, a lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number. IMPORTANCE It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846), we demonstrate herein that viral genome is indeed amplified in an E1- and E2-dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV life cycle using mutational approaches.

Investigation of the activity of baicalein towards Zika virus

BackgroundZika virus (ZIKV) is a mosquito transmitted virus spread primarily by Aedes species mosquitoes that can cause disease in humans, particularly when infection occurs in pregnancy where the virus can have a significant impact on the developing fetus. Despite this, there remains no prophylactic agent or therapeutic treatment for infection. Baicalein is a trihydroxyflavone, that is found in some traditional medicines commonly used in Asia, and has been shown to have several activities including antiviral properties. Importantly, studies have shown baicalein to be safe and well tolerated in humans, increasing its potential utilization.MethodsThis study sought to determine the anti-ZIKV activity of baicalein using a human cell line (A549). Cytotoxicity of baicalein was determined by the MTT assay, and the effect on ZIKV infection determined by treating A549 cells with baicalien at different time points in the infection process. Parameters including level of infection, virus production, viral protein expression and genome copy number were assessed by flow cytometry, plaque assay, western blot and quantitative RT-PCR, respectively.ResultsThe results showed that baicalein had a half-maximal cytotoxic concentration (CC50) of > 800 µM, and a half-maximal effective concentration (EC50) of 124.88 µM. Time-of-addition analysis showed that baicalein had an inhibitory effect on ZIKV infection at the adsorption and post-adsorption stages. Moreover, baicalein also exerted a significant viral inactivation activity on ZIKV (as well as on dengue virus and Japanese encephalitis virus) virions.ConclusionBaicalein has now been shown to possess anti-ZIKV activity in a human cell line.

Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.