DNA Polymerase Gene Research Articles

Validation and implementation of an orthopoxvirus qualitative real-time PCR for the diagnosis of monkeypox in the clinical laboratory.

The globally concerning outbreak of monkeypox virus (MPXV) in non-endemic countries in 2022 warranted an increased capacity of diagnostic clinical testing. In this study, we detail the Johns Hopkins analytical validation of a qualitative orthopoxvirus real-time PCR and characterize its analytical performance for MPXV testing. We also describe our first month of MPXV clinical laboratory diagnosis, including assay utilization and positivity. The analytical performance of a previously characterized orthopoxvirus assay that targets the DNA polymerase gene of non-variola orthopoxviruses was determined. In silico inclusivity analysis was performed for the primer and probe sequences. The limit of detection, reproducibility, agreement, and specificity were evaluated as well as target stability at room temperature. Clinical chart reviews were performed for patients who received testing within the first month of offering the assay for diagnosis. Alignment of the primer and probe binding regions of 151 sequenced genomes of MPXV from 2022 showed Sequence homology of 100%. The assay was able to detect MPXV at a concentration of at least 100 copies/mL and showed no cross reactivity with other organisms tested. Targets were stable for at least one week at room temperature. During the first month of implementation, a total of 33 patients were tested, 11 of whom were positive and presented mainly with rash in the genital region. Increased availability of MPXV testing is essential with the progressive increase in the number of cases. The non-variola orthopoxvirus assay is a sensitive and reproducible method for diagnosing monkeypox during the current surge in cases.

Characterization of DNA Polymerase Genes in Amazonian Amerindian Populations.

Due to their continuing geographic isolation, the Amerindian populations of the Brazilian Amazon present a different genetic profile when compared to other continental populations. Few studies have investigated genetic variants present in these populations, especially in the context of next-generation sequencing. Knowledge of the molecular profile of a population is one of the bases for inferences about human evolutionary history, in addition, it has the ability to assist in the validation of molecular biomarkers of susceptibility to complex and rare diseases, and in the improvement of specific precision medicine protocols applied to these populations and to populations with high Amerindian ancestry, such as Brazilians. DNA polymerases play essential roles in DNA replication, repair, recombination, or damage repair, and their influence on various clinical phenotypes has been demonstrated in the specialized literature. Thus, the aim of this study is to characterize the molecular profile of POLA1, POLE, POLG, POLQ, and REV3L genes in Amerindian populations from the Brazilian Amazon, comparing these findings with genomic data from five continental populations described in the gnomAD database, and with data from the Brazilian population described in ABraOM. We performed the whole exome sequencing (WES) of 63 Indigenous individuals. Our study described for the first time the allele frequency of 45 variants already described in the other continental populations, but never before described in the investigated Amerindian populations. Our results also describe eight unique variants of the investigated Amerindians populations, with predictions of moderate, modifier and high clinical impact. Our findings demonstrate the unique genetic profile of the Indigenous population of the Brazilian Amazon, reinforcing the need for further studies on these populations, and may contribute to the creation of public policies that optimize not only the quality of life of this population, but also of the Brazilian population.

Partial genetic characterisation of a novel alloherpesvirus detected by PCR in a farmed wels catfish (Silurus glanis).

By a broad-range PCR, we detected a novel herpesvirus (HV) in the specimen of a wels catfish (Silurus glanis) presenting disseminated, carp pox-like dermal lesions all over its body. The sequence analysis of the 463-bp PCR product from the viral DNA polymerase gene indicated the presence of a hitherto unknown virus, a putative member of the family Alloherpesviridae in the sample. Another PCR, targeting the terminase gene of fish HVs, provided an additional genomic fragment of over 1,000 bp. Surprisingly, the sequence of a co-amplified, off-target PCR product revealed its origin from a putative gene homologous to ORF87 and ORF45 of cyprinid HVs and anguillid herpesvirus 1 (AngHV-1), respectively. With specific primers, designed according to the genomic maps of the cyprinid and anguillid HVs, a genomic fragment of 15 kb was also amplified and sequenced by primer walking. In phylogeny inferences, based on several genes, the putative wels catfish HV clustered closest to various cyprinid HVs or to AngHV-1. The novel virus, named as silurid herpesvirus 2, represents a distinct species in the genus Cyprinivirus. However, its association with the skin disease remains unclear.

Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla.

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Detection of the peptidyl epitope for vaccine development against MPV

BackgroundMonkeypox Virus (MPV) is the cause of zoonotic disease characterized by skin-eruption with pus cell formation and lymphadenopathy. This virus belongs to the Orthopoxvirus genus with DNA as its genetic material. Previously, this infection was reported from Africa and occasionally from USA and UK. However, recently there is a sudden surge of infection in non-epidemic countries and a new strain of MPVhas been discovered. Therefore it is important to revisit the phylogeny of MPV with the addition of new strains. Recently WHO also stressed the need of developing vaccines for new strains. In this scenario we have two objectives for this study -first, to reveal the exact phylogenetic position of the 2022 strain and second, to identify specific peptides which may be used for vaccine development in the future. MethodsThe phylogenetic analysis was done with the help of Bayesian phylogeny. The dN/dS calculation was performed based on DNA polymerase genes of selected MPV strains. The peptidyl-epitope was searched in MPV2022/2 SLO strain with the help of several algorithms implemented in Allergen FP v.1.0, NetMHCII 2.3, MHCpred and Toxin Pred. The structure prediction of the proteins and peptides was performed through Hpepdock. The quality of the structures was validated through the Ramachandran plot. The molecular dynamics and simulation were performed through Gromacs software. The interaction between peptide and protein was assessed through Ligplot software. ResultsThe phylogenetic analysis revealed that the considered 2022 MPVstrains were close to the USA strains. The evolutionary analysis showed the volatile nature of the genome. Moreover, 9-mer peptide sequence was identified as an epitope for vaccine development. ConclusionsThe emergence of more virulent strains in near future may not be ruled out. Immunocompromised patients are more susceptible to this virus hence sub-unit vaccine is a better choice than a recombinant or attenuated vaccine against monkeypox. We have identified a small stretch of specific peptide which may be used for developing a subunit vaccine against this virus.

First Isolation of a Herpesvirus (Family Alloherpesviridae) from Great Lakes Lake Sturgeon (Acipenser fulvescens).

The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers’ postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.

Abundant and cosmopolitan lineage of cyanopodoviruses lacking a DNA polymerase gene

Cyanopodoviruses affect the mortality and population dynamics of the unicellular picocyanobacteria Prochlorococcus and Synechococcus, the dominant primary producers in the oceans. Known cyanopodoviruses all contain the DNA polymerase gene (DNA pol) that is important for phage DNA replication and widely used in field quantification and diversity studies. However, we isolated 18 cyanopodoviruses without identifiable DNA pol. They form a new MPP-C clade that was separated from the existing MPP-A, MPP-B, and P-RSP2 clades. The MPP-C phages have the smallest genomes (37.3–37.9 kb) among sequenced cyanophages, and show longer latent periods than the MPP-B phages. Metagenomic reads of both clades are highly abundant in surface waters, but the MPP-C phages show higher relative abundance in surface waters than in deeper waters, while MPP-B phages have higher relative abundance in deeper waters. Our study reveals that cyanophages with distinct genomic contents and infection kinetics can exhibit different depth profiles in the oceans.

Screening of West Nile Virus, Herpesvirus, and Parvovirus in Rattus spp. in Klang Valley, Malaysia

Free-roaming and scavenging lifestyles of Rattus spp. in densely populated urban areas expose them to multiple pathogens that facilitate the transmission of infection to the human population more rapidly, raising public health concerns. There is limited information on the status of rat susceptibility to virus infection, particularly West Nile virus (WNV), herpesvirus, and parvovirus, to prepare for emerging zoonosis. A total of 23 (n = 23) blood samples collected from Rattus spp. in the wet market areas of Klang Valley, Malaysia, were subjected to molecular assay using a one-step reverse transcription-polymerase chain reaction (RT-PCR) to detect the highly conserved region of the WNV capsid and pre-membrane protein via nested polymerase chain reaction (PCR) assay targeting highly conserved amino acid motifs within the herpesviral DNA-directed DNA polymerase gene (DPOL) and polymerase chain reaction (PCR) assay targeting the parvovirus non-structural (NS) protein. As a result, 4 out of 23 (17.39%) rats were positive for herpesvirus DNA, but none were positive for WNV RNA and parvovirus DNA. The positive PCR amplicons of herpesvirus DNA were subjected to partial DNA sequencing analysis, 100% identical to Acomys herpesvirus SVMS 226,222 from Betaherpesvirinae, which is highly suggestive of rat cytomegalovirus (RCMV). This study has successfully demonstrated the presence of RCMV from Rattus spp. in the Klang Valley. The RCMV potentially crosses species barriers and establishes infection, raising public health concerns. The non-viraemic state of WNV or parvovirus infection, low sample size, and limited niche distribution emphasise the need for the expansion of this study in the future.

Isoferulic acid affords the antiviral potential and restrains white spot syndrome virus proliferation in crayfish (Procambarus clarkii)

White spot syndrome virus (WSSV), the causative agent of White spot disease (WSD), not only caused huge economic losses, but also greatly limited the development of crustacean breeding industry. Chinese herbs are widely used and have great application potential in antiviral research. In thist study, the antiviral effects of 11 Chinese herb extracts were evaluated in Procambarus clarkii (crayfish) model of WSSV infection, among which Cimicifuga foetida L. extract showed the highest inhibition rate (88.22%) against WSSV replication. On this basis, the anti-WSSV effect of Isoferulic acid (IFA), a major bioactive component of Cimicifuga foetida L., was further investigated. The results showed that IFA significantly inhibited WSSV replication (96.57%, 50 mg/kg) in a dose-dependent manner and significantly improved the survival of WSSV-challenged crayfish. IFA had the potential preventive and therapeutic effect against WSSV infection, and the preventive effect was better than the therapeutic effect. Mechanistically, IFA suppressed the expression of the immediate early gene ie1, the DNA polymerase gene DNApol and envelope protein gene Vp28 to inhibit WSSV replication, activated TLR, CTL and ProPo genes expression to block ie1 transcription, and suppressed BAF, STAT, NF-κb and Crustin 1 genes expression to elevate innate immunity against WSSV infection. Besides, IFA upregulated the cMnSOD, mMnSOD, CAT and GST genes expression, and downregulated the COX-1 and COX-2 genes expression to balance the oxidative stress and inflammatory damage caused by WSSV infection. This study provides a theoretical basis and meaningful reference for the development of IFA as a highly effective and practical anti-WSSV drug.

Novel adenoviruses from captive psittacine birds in Slovenia

To assess the prevalence of adenoviruses in psittacine birds kept in Slovenia, 258 cloacal swabs were collected from different psittacine species and screened by a nested PCR with degenerate, consensus primers targeting the adenoviral DNA polymerase gene. Forty-two samples were found to be positive. By sequencing, 28 samples from 10 different parrot species were identified as the formerly described siadenovirus, psittacine adenovirus 2 (PsAdV-2). A second siadenovirus, a variant of PsAdV-5 (described earlier from Pacific parrotlet, sun parakeet, cockatiel and budgerigar) was found in seven budgerigars, two cockatiels and an amazon parrot species. A variant of Meyer’s parrot adenovirus (aviadenovirus, proposed PsAdV-8) was identified in an African grey parrot and a cockatiel. Two novel atadenoviruses were revealed in cockatiel (PsAdV-9) and rose-ringed parakeet (PsAdV-10). These results support the earlier finding that many PsAdVs can cross the species barrier among psittacines, especially effectively in the case of PsAdV-2.

Infection with a Recently Discovered Gammaherpesvirus Variant in European Badgers, Meles meles, is Associated with Higher Relative Viral Loads in Blood.

Herpesviruses are ubiquitous pathogens infecting most animals. Although host immunity continually coevolves to combat virulence, viral variants with enhanced transmissibility or virulence occasionally emerge, resulting in disease burdens in host populations. Mustelid gammaherpesvirus 1 (MusGHV-1) is the only herpesvirus species identified thus far in European badgers, Meles meles. No MusGHV-1 associated pathomorbidity has been reported, but reactivation of MusGHV-1 in genital tracts is linked to impaired female reproductive success. An analysis of a short sequence from the highly conserved DNA polymerase (DNApol) gene previously identified two variants in a single host population. Here we compared genetic variance in blood samples from 66 known individuals of this same free-ranging badger population using a partial sequence comprising 2874 nucleotides of the DNApol gene, among which we identified 15 nucleotide differences resulting in 5 amino acid differences. Prevalence was 86% (59/66) for the common and 17% (11/66) for the novel variant, with 6% (4/66) of badgers presenting with coinfection. MusGHV-1 variants were distributed unevenly across the population, with individuals infected with the novel genotype clustered in 3 of 25 contiguous social groups. Individuals infected with the novel variant had significantly higher MusGHV-1 viral loads in their blood (p = 0.002) after adjusting for age (juveniles > adults, p < 0.001) and season (summer > spring and autumn, p = 0.005; mixed-effect linear regression), likely indicating higher virulence of the novel variant. Further genome-wide analyses of MusGHV-1 host resistance genes and host phenotypic variations are required to clarify the drivers and sequelae of this new MusGHV-1 variant.

Simian adenoviruses: Molecular and serological survey in monkeys and humans in Thailand

Simian adenoviruses are in the genus Mastadenovirus of the family Adenoviridae. This family is composed of non-enveloped, double-stranded DNA viruses that infect a wide range of animals. Mastadenoviruses infect mammals, including non-human primates and humans. The close genetic relatedness between simian and human adenoviruses, with its associated potential for the cross-species transmission of zoonotic adenoviruses from monkeys to humans and vice versa, poses important health concerns and thus warrants research. In this study, we performed a molecular survey of adenoviruses in monkeys in Thailand. Most of the monkeys tested here were long-tailed macaques, free-ranging in areas close to human territories across four provinces: Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan. A few fecal samples from captive wild monkeys (a stump-tailed macaque, pig-tailed macaques, gibbons, and a leaf monkey) were also tested. Adenoviruses were detected in 33.3% (70 out of 210) of the fecal or rectal swab samples. The viruses identified in these samples included Simian adenovirus (SAdV)-A, SAdV-B, SAdV-H, Human adenovirus (HAdV)-D, HAdV-G, and a bat adenovirus species. One SAdV-B, SAdV RBR-7-10, was isolated from a long-tailed macaque fecal sample and identified by mass spectrometry. Its full hexon gene and nearly complete DNA polymerase gene were sequenced and analyzed, and the virions were imaged by transmission electron microscopy. The SAdV RBR-7-10 virus was used in a microneutralization assay to identify virus-specific antibodies in monkey plasma and human serum samples collected from the same areas in Prachuap Khiri Khan Province. We detected neutralizing antibodies against SAdV RBR-7-10 in 6.8% (n = 103) of the monkey samples but in none of the 125 human serum samples, suggesting no cross-species transmission of SAdV RBR-7-10 occurred at this study site. Nevertheless, a continuing surveillance of pathogens in monkeys is warranted to quickly identify possible emerging zoonotic outbreaks.

Heterogeneity and viral replication fitness of HSV-1 clinical isolates with mutations in the thymidine kinase and DNA polymerase.

Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge. To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness. Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays. Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to. The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure.

Detection of Cetacean Poxvirus in Peruvian Common Bottlenose Dolphins (Tursiops truncatus) Using a Pan-Poxvirus PCR.

Cetacean poxviruses (CePVs) cause ‘tattoo’ skin lesions in small and large cetaceans worldwide. Although the disease has been known for decades, genomic data for these poxviruses are very limited, with the exception of CePV-Tursiops aduncus, which was completely sequenced in 2020. Using a newly developed pan-pox real-time PCR system targeting a conserved nucleotide sequence located within the Monkeypox virus D6R gene, we rapidly detected the CePV genome in typical skin lesions collected from two Peruvian common bottlenose dolphins (Tursiops truncatus) by-caught off Peru in 1993. Phylogenetic analyses based on the sequencing of the DNA polymerase and DNA topoisomerase genes showed that the two viruses are very closely related to each other, although the dolphins they infected pertained to different ecotypes. The poxviruses described in this study belong to CePV-1, a heterogeneous clade that infects many species of dolphins (Delphinidae) and porpoises (Phocoenidae). Among this clade, the T. truncatus CePVs from Peru were more related to the viruses infecting Delphinidae than to those detected in Phocoenidae. This is the first time that CePVs were identified in free-ranging odontocetes from the Eastern Pacific, surprisingly in 30-year-old samples. These data further suggest a close and long-standing pathogen–host co-evolution, resulting in different lineages of CePVs.

Delineating selective vulnerability of inhibitory interneurons in Alpers' syndrome.

AimsAlpers' syndrome is a severe neurodegenerative disease typically caused by bi‐allelic variants in the mitochondrial DNA (mtDNA) polymerase gene, POLG, leading to mtDNA depletion. Intractable epilepsy, often with an occipital focus, and extensive neurodegeneration are prominent features of Alpers' syndrome. Mitochondrial oxidative phosphorylation (OXPHOS) is severely impaired with mtDNA depletion and is likely to be a major contributor to the epilepsy and neurodegeneration in Alpers' syndrome. We hypothesised that parvalbumin‐positive(+) interneurons, a neuronal class critical for inhibitory regulation of physiological cortical rhythms, would be particularly vulnerable in Alpers' syndrome due to the excessive energy demands necessary to sustain their fast‐spiking activity.MethodsWe performed a quantitative neuropathological investigation of inhibitory interneuron subtypes (parvalbumin+, calretinin+, calbindin+, somatostatin interneurons+) in postmortem neocortex from 14 Alpers' syndrome patients, five sudden unexpected death in epilepsy (SUDEP) patients (to control for effects of epilepsy) and nine controls.ResultsWe identified a severe loss of parvalbumin+ interneurons and clear evidence of OXPHOS impairment in those that remained. Comparison of regional abundance of interneuron subtypes in control tissues demonstrated enrichment of parvalbumin+ interneurons in the occipital cortex, while other subtypes did not exhibit such topographic specificity.ConclusionsThese findings suggest that the vulnerability of parvalbumin+ interneurons to OXPHOS deficits coupled with the high abundance of parvalbumin+ interneurons in the occipital cortex is a key factor in the aetiology of the occipital‐predominant epilepsy that characterises Alpers' syndrome. These findings provide novel insights into Alpers' syndrome neuropathology, with important implications for the development of preclinical models and disease‐modifying therapeutics.

Detection of herpesviral hematopoietic necrosis virus (Cyprinid herpesvirus 2, CyHV-2) from goldfish, Carassius auratus (L.) in Korea

In April and June 2014, mortalities of goldfish occurred at Korea. The principal signs included pale gills, severely enlarged and softened spleen and kidney and red liver. Moreover, the histopathological characteristics observed mainly in hematopoietic tissues of kidney, gill lamellae and intestine. The predominant histopathological changes were severe necrosis of hematopoietic tissue. In addition, nucleus exhibiting peripherally displaced chromatin were particularly abundant in the kidney of affected fish. The histological examination led to hypothesize the implication of a virus in the mortality. The hematopoietic necrosis herpes-virus (Cyprinid herpesvirus 2, CyHV-2) DNA polymerase gene successfully detected in DNA extracted from kidney and spleen of affected fish using PCR assay and showed 100% identity with already deposited CyHV-2 DNA polymerase gene in NCBI. Artificial infection trials using affected tissues filtrates gave cumulative mortalities of 30% for virus injected goldfish. In the present study, CyHV-2 was detected and identified as the causative pathogen of the epizootic in goldfish in Korea.

Adenoviral infection in 5 red-tailed hawks and a broad-winged hawk.

Adenoviral infections among raptors are best described in falcons and are characterized most commonly by necrotizing hepatitis and splenitis; only one case has been reported in a hawk. Five red-tailed hawks (Buteo jamaicensis) and a broad-winged hawk (Buteo platypterus) had an adenoviral infection based on history, histopathology, negative-stain electron microscopy, and PCR. All birds had acute onset of illness resulting in death; 3 had evidence of a concurrent bacterial infection. Microscopically, all 6 birds had solitary, pale eosinophilic-to-amphophilic, intranuclear inclusion bodies within presumed hematopoietic cells in bone marrow and macrophages in spleen. Five of the 6 birds had similar inclusions within hepatocytes and Kupffer cells. All but one bird had severe bone marrow necrosis. There was moderate splenic necrosis (3 of 6) and mild-to-marked hepatic necrosis (4 of 6). Negative-stain electron microscopy demonstrated adenoviral particles in bone marrow (5 of 6), liver (1 of 5), and/or spleen (1 of 5). PCR was positive for adenovirus in bone marrow (3 of 5), liver (1 of 3), spleen (4 of 6), and/or intestinal contents (2 of 3). Viral DNA polymerase gene sequences clustered within the Siadenovirus genus. There was 99% nucleotide identity to one another and 90% nucleotide identity with the closest related adenovirus (Harris hawk, EU715130). Our case series expands on the limited knowledge of adenoviral infections in hawks. The splenic and hepatic necrosis, and particularly the hitherto unreported bone marrow necrosis, suggest that adenoviral infection is clinically relevant and potentially fatal in hawks.

The mitochondrial genome of a wild edible mushroom, Russula rosea

Russula rosea is a common wild edible ectomycorrhizal fungus, which is widely distributed all over the world. We assembled the complete mitochondrial genome of R. rosea with the total length was 54177 bp and the GC content of 22.34%. It contains a total of 57 genes, including 14 standard protein-coding genes, one conserved ribosomal protein S3 gene (rps3), two rRNA genes, 24 tRNA genes, 15 free-standing open reading frames (ORFs) and one DNA polymerase gene (dpo). Mitochondrial genome found a close evolutionary relationship between Russula rosea and Russula lepida, which was helpful to study the genetic evolutionary relationship of edible fungi.

The Nuclear DNA Sensor IFI16 Indiscriminately Binds to and Diminishes Accessibility of the HSV-1 Genome to Suppress Infection.

ABSTRACTHuman cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, including DNA sensors. The interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor that recognizes double-stranded DNA from a number of viral sources, including genomes of nuclear-replicating viruses. Among these is the prevalent human pathogen herpes simplex virus 1 (HSV-1). Upon binding to the HSV-1 DNA genome, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we used a multiomics approach of DNA sequencing techniques paired with targeted mass spectrometry to obtain an extensive view of the interaction between IFI16 and the HSV-1 genome and how this binding affects the viral DNA structure and protein expression. Through chromatin immunoaffinity purification coupled with next-generation DNA sequencing (ChIP-seq), we found that IFI16 binds to the HSV-1 genome in a sequence-independent manner while simultaneously exhibiting broad enrichment at two loci: UL30, the viral DNA polymerase gene, and US1 to US7. The assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed that these two regions are among the most accessible stretches of DNA on the genome, thereby facilitating IFI16 binding. Accessibility of the entire HSV-1 genome is elevated upon IFI16 knockout, indicating that expression of IFI16 globally induces chromatinization of viral DNA. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry of viral proteins representing 80% of the HSV-1 genome. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, coordinating epigenetic silencing of the viral genome and decreasing protein expression and virus replication.IMPORTANCE Mammalian host defense against viral infection includes broad-acting cellular restriction factors, as well as effectors of intrinsic and innate immunity. IFI16 is a critical nuclear host defense factor and intrinsic immune protein involved in binding viral DNA genomes, thereby repressing the replication of nucleus-replicating viruses, including the human herpes simplex virus 1. What has remained unclear is where on the viral genome IFI16 binds and how binding affects both viral DNA structural accessibility and viral protein expression. Our study provides a global view of where and how a nuclear restriction factor of DNA viruses associates with viral genomes to exert antiviral functions during early stages of an acute virus infection. Our study can additionally serve as a systems-level model to evaluate nuclear DNA sensor interactions with viral genomes, as well as the antiviral outcomes of transcriptionally silencing pathogen-derived DNA.

Outbreaks of Avipoxvirus Clade E in Vaccinated Broiler Breeders with Exacerbated Beak Injuries and Sex Differences in Severity.

Avipoxvirus affects chickens and wild birds, and it is characterized by lesions on the nonfeathered parts of the body (the cutaneous form), or necrotic lesions in the upper respiratory tract (the diphtheritic form). In poultry farming, avian pox is usually controlled by live attenuated vaccines. However, there have been many reports of outbreaks, even in flocks of vaccinated birds. In the present study, different outbreaks of the emerging clade E avipoxvirus were detected in commercial breeder flocks of chickens vaccinated against fowlpox virus in Southeast Brazil. Clinical manifestations of these outbreaks included a marked prevalence of moderate to severe progressive lesions in the beaks of affected birds, especially in roosters with increased mortality (up to 8.48%). Also, a reduced hatchability (up to 20.77% fewer hatching eggs) was observed in these flocks. Analysis of clinical samples through light and transmission electron microscopy revealed the presence of Bollinger bodies and poxvirus particles in epithelial cells and affecting chondrocytes. PCR, sequencing, and phylogenetic analysis of major core protein (P4b) and DNA polymerase (pol) genes identified this virus as clade E avipoxvirus. We also developed qPCR assays for open reading frames (ORFs) 49, 114, and 159 to detect and quantify this emergent virus. These results show the arrival and initial spread of this pathogen in the poultry industry, which was associated with harmful outbreaks and exacerbated clinical manifestations in vaccinated commercial breeder flocks. This study also highlights the relevance of permanent vigilance and the need to improve sanitary and vaccination programs.