Research

10 pages, 4360 KiB

Open AccessCommunication

Preservation of scRNA-Seq Libraries Using Existing Inactivation Protocols

by Gail L. Sturdevant, Kimberly D. Meade-White, Sonja M. Best and Emily Speranza

Pathogens 2024, 13(2), 167; https://doi.org/10.3390/pathogens13020167 - 13 Feb 2024

Cited by 1 | Viewed by 1172

Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this [...] Read more.

Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this analysis in high containment settings such as biosafety level 3 (BSL-3), BSL-3+ and BSL-4. Working in containment is necessary for many important pathogens, such as Ebola virus, Marburg virus, Lassa virus, Nipah and Hendra viruses. Since standard operating procedures (SOPs) for inactivation are extensive and may compromise sample integrity, we tested whether the removal of single-cell sequencing libraries from containment laboratories using existing inactivation protocols for nucleic acid extraction (Trizol, RLT buffer, or AVL buffer) was feasible. We have demonstrated that the inactivation does not affect sample quality and can work with existing methods for inactivation. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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21 pages, 2534 KiB

Open AccessFeature PaperArticle

Five Species of Wild Freshwater Sport Fish in Wisconsin, USA, Reveal Highly Diverse Viromes

by Charlotte E. Ford, Christopher D. Dunn, Eric M. Leis, Whitney A. Thiel and Tony L. Goldberg

Pathogens 2024, 13(2), 150; https://doi.org/10.3390/pathogens13020150 - 07 Feb 2024

Viewed by 1226

Abstract

Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of [...] Read more.

Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of freshwater fish in Wisconsin, USA: bluegill (Lepomis macrochirus), brown trout (Salmo trutta), lake sturgeon (Acipenser fulvescens), northern pike (Esox lucius), and walleye (Sander vitreus). We analyzed 103 blood serum samples collected during a state-wide survey from 2016 to 2020 and used a metagenomic approach for virus detection to identify known and previously uncharacterized virus sequences. We then characterized viruses phylogenetically and quantified prevalence, richness, and relative abundance for each virus. Within these viromes, we identified 19 viruses from 11 viral families: Amnoonviridae, Circoviridae, Coronaviridae, Hepadnaviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Matonaviridae, Narnaviridae, Nudnaviridae, and Spinareoviridae, 17 of which were previously undescribed. Among these viruses was the first fish-associated coronavirus from the Gammacoronavirus genus, which was present in 11/15 (73%) of S. vitreus. These results demonstrate that, similar to marine fish, freshwater fish also harbor diverse relatives of viruses important to the health of fish and other animals, although it currently remains unknown what effect, if any, the viruses we identified may have on fish health. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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12 pages, 1340 KiB

Open AccessArticle

Comprehensive Analysis of HIV-1 Integrase Resistance-Related Mutations in African Countries

by Francesco Branda, Marta Giovanetti, Leonardo Sernicola, Stefania Farcomeni, Massimo Ciccozzi and Alessandra Borsetti

Pathogens 2024, 13(2), 102; https://doi.org/10.3390/pathogens13020102 - 24 Jan 2024

Viewed by 903

Abstract

The growing emergence of non-nucleoside reverse transcriptase inhibitor (NNRTI) HIV drug resistance in sub-Saharan Africa (SSA) led to the World Health Organization (WHO) recommending, in 2018, a transition to dolutegravir (DTG) as a first-line antiretroviral therapy (ART) in SSA. The broad HIV-1 genetic [...] Read more.

The growing emergence of non-nucleoside reverse transcriptase inhibitor (NNRTI) HIV drug resistance in sub-Saharan Africa (SSA) led to the World Health Organization (WHO) recommending, in 2018, a transition to dolutegravir (DTG) as a first-line antiretroviral therapy (ART) in SSA. The broad HIV-1 genetic diversity in SSA could shape DTG effectiveness and the pattern of drug resistance mutations (DRMs) in this region. This study evaluated HIV-1 integrase (IN) DRMs and conserved regions among published groups M, N, O, and P HIV-1 sequences spanning forty years of the HIV epidemic during the transition of DTG-based ART. Overall, we found low levels of integrase strand transfer inhibitor (INSTI)-DRMs (<1%) across HIV groups between the years 1983 and 2023; however, it was unexpected to detect DRMs at statistically significantly higher frequencies in pre-INSTI (1983–2007) than in the INSTI (2008–2023) era. The variability of accessory INSTI-DRMs depended on the HIV subtypes, with implications for susceptibility to DTG. Our findings provide new perspectives on the molecular epidemiology and drug resistance profiles of INSTIs in SSA, emphasizing the need for ongoing surveillance and customized treatment approaches to address the continent’s varied HIV subtypes and changing resistance patterns. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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17 pages, 4501 KiB

Open AccessArticle

Multivalent Epigraph Hemagglutinin Vaccine Protects against Influenza B Virus in Mice

by Erika Petro-Turnquist, Brigette Corder Kampfe, Amber Gadeken, Matthew J. Pekarek and Eric A. Weaver

Pathogens 2024, 13(2), 97; https://doi.org/10.3390/pathogens13020097 - 23 Jan 2024

Viewed by 1185

Abstract

Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only [...] Read more.

Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only induce strain-specific responses and have suboptimal efficacy when mismatched from circulating strains. Further, two influenza B virus lineages have been described, B/Yamagata-like and B/Victoria-like, and the limited cross-reactivity between the two lineages provides an additional barrier in developing a universal influenza B virus vaccine. Here, we report a novel multivalent vaccine using computationally designed Epigraph hemagglutinin proteins targeting both the B/Yamagata-like and B/Victoria-like lineages. When compared to the quadrivalent commercial vaccine, the Epigraph vaccine demonstrated increased breadth of neutralizing antibody and T cell responses. After lethal heterologous influenza B virus challenge, mice immunized with the Epigraph vaccine were completely protected against both weight loss and mortality. The superior cross-reactive immunity conferred by the Epigraph vaccine immunogens supports their continued investigation as a universal influenza B virus vaccine. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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13 pages, 1704 KiB

Open AccessArticle

Complement Suppresses the Initial Type 1 Interferon Response to Ocular Herpes Simplex Virus Type 1 Infection in Mice

by Daniel J. J. Carr, Adrian Filiberti and Grzegorz B. Gmyrek

Pathogens 2024, 13(1), 74; https://doi.org/10.3390/pathogens13010074 - 13 Jan 2024

Viewed by 1328

Abstract

The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, [...] Read more.

The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey’s post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-β and downstream effector genes including tetherin and RNase L (p < 0.05, Mann–Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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17 pages, 4577 KiB

Open AccessFeature PaperArticle

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality

by Adam J. Hume, Judith Olejnik, Mitchell R. White, Jessie Huang, Jacquelyn Turcinovic, Baylee Heiden, Pushpinder S. Bawa, Christopher J. Williams, Nickolas G. Gorham, Yuriy O. Alekseyev, John H. Connor, Darrell N. Kotton and Elke Mühlberger

Pathogens 2024, 13(1), 62; https://doi.org/10.3390/pathogens13010062 - 09 Jan 2024

Cited by 1 | Viewed by 1892

Abstract

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) [...] Read more.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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9 pages, 1120 KiB

Open AccessCommunication

An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants

by Petros Bozidis, Eleni Petridi and Konstantina Gartzonika

Pathogens 2023, 12(8), 1017; https://doi.org/10.3390/pathogens12081017 - 06 Aug 2023

Viewed by 1002

Abstract

As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, [...] Read more.

As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, BA.2, BA.3, BA.4, and BA.5. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) that will be able to distinguish the basic sub-variants of Omicron in a rapid and specific way. Full genome sequences of Omicron strains with high frequency and wide geographical distribution were retrieved by the NCBI Virus and ENA databases. These sequences were compared to each other in order to locate single nucleotide polymorphisms common to all strains of the same sub-variant. These polymorphisms should also be capable of distinguishing Omicron sub-variants not only from each other but from previously circulating variants of SARS-CoV-2 as well. Thus, specific primers targeting characteristic polymorphisms of the four Omicron main branches BA.1, BA.2, BA.4, and BA.5 were designed according to the principles of the amplification refractory mutation system (ARMS) and with the ability to react under multiplex PCR conditions. According to our results, the ARMS-multiplex PCR could successfully distinguish all Omicron sub-variants that carry the corresponding mutations. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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22 pages, 13797 KiB

Open AccessEditor’s ChoiceArticle

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

by Judith Olejnik, Adam J. Hume, Stephen J. Ross, Whitney A. Scoon, Scott Seitz, Mitchell R. White, Ben Slutzky, Nadezhda E. Yun and Elke Mühlberger

Pathogens 2023, 12(7), 952; https://doi.org/10.3390/pathogens12070952 - 19 Jul 2023

Cited by 2 | Viewed by 2734

Abstract

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the [...] Read more.

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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14 pages, 5257 KiB

Open AccessArticle

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

by Judith Olejnik, Juliette Leon, Daniel Michelson, Kaitavjeet Chowdhary, Silvia Galvan-Pena, Christophe Benoist, Elke Mühlberger and Adam J. Hume

Pathogens 2023, 12(2), 342; https://doi.org/10.3390/pathogens12020342 - 17 Feb 2023

Cited by 3 | Viewed by 1608

Abstract

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, [...] Read more.

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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12 pages, 1043 KiB

Open AccessArticle

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021

by Marta Falcão, Margarida Barros, Margarida D. Duarte, Fábio Abade dos Santos, Teresa Fagulha, Margarida Henriques, Fernanda Ramos, Ana Duarte, Tiago Luís, Ricardo Parreira and Sílvia C. Barros

Pathogens 2023, 12(2), 150; https://doi.org/10.3390/pathogens12020150 - 17 Jan 2023

Cited by 2 | Viewed by 2083

Abstract

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo [...] Read more.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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