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Application of Advanced Molecular Methods to Study Infections

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 24357

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Prof. Dr. Elisabetta Tanzi

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Department of Health Sciences, University of Milan, Via Pascal 36, 20133 Milan, Italy
Interests: molecular epidemiology; phylogenetic analysis; molecular diagnostics; sexually transmitted infections; infections and cancer; emerging infections
Special Issues, Collections and Topics in MDPI journals

Dr. Marta Canuti

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Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milan, Italy
Interests: virus discovery; virus ecology; viral transmission dynamics; parvovirus; virus evolution
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Dear Colleagues,

In recent decades, several advancements in molecular laboratory techniques, sequencing platforms, and sequence analysis tools have revolutionized the way we diagnose and study infections. Such methods can be used to study several different characteristics of infectious agents, from ecology and epidemiology to molecular biology, evolution, and pathogenicity, as well as to investigate important aspects linked to infectious diseases, such as outbreak tracing or control and prevention.

This Special Issue aims to collect research contributions or review articles that involve the description, development, or use of state-of-the-art or new molecular or sequence analyses methods to explore the world of infections and infectious microorganisms at a molecular level. Contributions both regarding pathogens that are relevant for human and animal health as well as microbes that are not pathogenic or whose pathogenic potential is still uncharacterized will be considered. Manuscripts including novel bioinformatic pipelines and comparative approaches that are related to clarifying molecular aspects of infectious agents are also welcome.

Prof. Dr. Elisabetta Tanzi
Dr. Marta Canuti
Guest Editors

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Keywords

molecular epidemiology
genotyping
novel screening and diagnostic methods
epidemic tracing through molecular characterization
next-generation sequencing
metagenomics
molecular ecology and evolution of microbes
molecular mechanisms of pathogenicity
mechanisms of pathogen-host interactions
infectious disease emergence
cross-species transmission
phylogeny
phylodynamics
microbiome/virome/infectome

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Published Papers (11 papers)

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16 pages, 1980 KiB

Open AccessArticle

Evaluation of the Virulence Potential of Listeria monocytogenes through the Characterization of the Truncated Forms of Internalin A

by Giulia Magagna, Maria Gori, Valeria Russini, Veronica De Angelis, Elisa Spinelli, Virginia Filipello, Vito Massimo Tranquillo, Maria Laura De Marchis, Teresa Bossù, Clara Fappani, Elisabetta Tanzi and Guido Finazzi

Int. J. Mol. Sci. 2023, 24(12), 10141; https://doi.org/10.3390/ijms241210141 - 14 Jun 2023

Cited by 4 | Viewed by 1741

Abstract

Listeria monocytogenes is a widespread Gram-positive pathogenic bacterium that causes listeriosis, a rather rare but severe foodborne disease. Pregnant women, infants, the elderly, and immunocompromised individuals are considered particularly at risk. L. monocytogenes can contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis. L. monocytogenes virulence factors include internalin A (InlA), a surface protein known to facilitate bacterial uptake by human intestinal epithelial cells that express the E-cadherin receptor. Previous studies have demonstrated that the presence of premature stop codon (PMSC) mutations naturally occurring in inlA lead to the production of a truncated protein correlated with attenuate virulence. In this study, 849 L. monocytogenes isolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in the inlA gene using Sanger sequencing or whole-genome sequencing (WGS). PMSC mutations were found in 27% of the isolates, predominantly in those belonging to hypovirulent clones (ST9 and ST121). The presence of inlA PMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential of L. monocytogenes circulating in Italy and could help to improve risk assessment approaches. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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13 pages, 1641 KiB

Open AccessArticle

Neutrophil Extracellular Traps and Platelet Activation for Identifying Severe Episodes and Clinical Trajectories in COVID-19

by Paula González-Jiménez, Raúl Méndez, Ana Latorre, Mónica Piqueras, María Nieves Balaguer-Cartagena, Antonio Moscardó, Ricardo Alonso, David Hervás, Soledad Reyes and Rosario Menéndez

Int. J. Mol. Sci. 2023, 24(7), 6690; https://doi.org/10.3390/ijms24076690 - 3 Apr 2023

Cited by 4 | Viewed by 1891

Abstract

The role of NETs and platelet activation in COVID-19 is scarcely known. We aimed to evaluate the role of NETs (citrullinated histone H3 [CitH3], cell-free DNA [cfDNA]) and platelet activation markers (soluble CD40 ligand [CD40L] and P-selectin) in estimating the hazard of different clinical trajectories in patients with COVID-19. We performed a prospective study of 204 patients, categorized as outpatient, hospitalized and ICU-admitted. A multistate model was designed to estimate probabilities of clinical transitions across varying states, such as emergency department (ED) visit, discharge (outpatient), ward admission, ICU admission and death. Levels of cfDNA, CitH3 and P-selectin were associated with the severity of presentation and analytical parameters. The model showed an increased risk of higher levels of CitH3 and P-selectin for ED-to-ICU transitions (Hazard Ratio [HR]: 1.35 and 1.31, respectively), as well as an elevated risk of higher levels of P-selectin for ward-to-death transitions (HR: 1.09). Elevated levels of CitH3 (HR: 0.90), cfDNA (HR: 0.84) and P-selectin (HR: 0.91) decreased the probability of ward-to-discharge transitions. A similar trend existed for elevated levels of P-selectin and ICU-to-ward transitions (HR 0.40); In conclusion, increased NET and P-selectin levels are associated with more severe episodes and can prove useful in estimating different clinical trajectories. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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21 pages, 3511 KiB

Open AccessArticle

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain

by Begoña Febrer-Sendra, Pedro Fernández-Soto, Juan García-Bernalt Diego, Beatriz Crego-Vicente, Anabel Negredo, Juan Luis Muñor-Bellido, Moncef Belhassen-García, María Paz Sánchez-Seco and Antonio Muro

Int. J. Mol. Sci. 2023, 24(7), 6411; https://doi.org/10.3390/ijms24076411 - 29 Mar 2023

Cited by 6 | Viewed by 2813

Abstract

Crimean–Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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12 pages, 1470 KiB

Open AccessArticle

Metabolomic and Proteomic Profiling of Porcine Intestinal Epithelial Cells Infected with Porcine Epidemic Diarrhea Virus

by Haifei Wang, Peng Hui, Yoshinobu Uemoto, Yueyun Ding, Zongjun Yin and Wenbin Bao

Int. J. Mol. Sci. 2023, 24(6), 5071; https://doi.org/10.3390/ijms24065071 - 7 Mar 2023

Cited by 5 | Viewed by 2240

Abstract

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers (p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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13 pages, 3022 KiB

Open AccessArticle

Establishment of a Pseudovirus Platform for Neuraminidase Inhibiting Antibody Analysis

by Yulia Desheva, Nadezhda Petkova, Igor Losev, Dmitry Guzhov, Alexey Go, Yu-Chan Chao and Chih-Hsuan Tsai

Int. J. Mol. Sci. 2023, 24(3), 2376; https://doi.org/10.3390/ijms24032376 - 25 Jan 2023

Cited by 1 | Viewed by 1954

Abstract

Neuraminidase (NA)-based immunity to influenza can be useful for protecting against novel antigenic variants. To develop safe and effective tools to assess NA-based immunity, we generated a baculovirus-based pseudotyped virus, N1-Bac, that expresses the full-length NA of the influenza A/California/07/2009 (H1N1)pdm09 strain. We evaluated the level of NA-inhibiting (NI) antibodies in the paired blood sera of influenza patients by means of an enzyme-linked lectin assay (ELLA) using the influenza virus or N1-Bac. Additionally, we evaluated the level of NA antibodies by means of the enzyme-linked immunosorbent assay (ELISA) with an N1-expressing Sf21 culture. We detected a strong correlation between our results from using the influenza virus and NA-Bac pseudoviruses to detect NI antibodies and a medium-strong correlation between NI antibodies and NA antibodies determined by an N1-cell ELISA, indicating that baculovirus-based platforms can be successfully used to evaluate NI or NA antibodies. Furthermore, animal experiments showed that immunization with N1-Bac protected against infection with a drift variant of the A/H1N1pdm09 influenza virus. Our results demonstrate that recombinant baculovirus can be an effective influenza pseudotype to evaluate influenza serologic immunity and protect against influenza virus infection. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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10 pages, 1351 KiB

Open AccessArticle

Evaluation of Human Papilloma Virus (HPV) Genotyping and Viral Load Determination as Diagnostic Biomarkers of Cervical Cancer Risk

by Marianna Martinelli, Chiara Giubbi, Laura Saderi, Rosario Musumeci, Federica Perdoni, Biagio Eugenio Leone, Robert Fruscio, Fabio Landoni, Andrea Piana, Giovanni Sotgiu and Clementina Elvezia Cocuzza

Int. J. Mol. Sci. 2023, 24(2), 1320; https://doi.org/10.3390/ijms24021320 - 10 Jan 2023

Cited by 2 | Viewed by 3106

Abstract

HPV testing in cervical cancer screening programs offers the possibility of introducing molecular standardized biomarkers for the triage of HPV-positive women. This study aimed to evaluate the role of HPV genotyping and viral load as possible diagnostic biomarkers of high-grade cervical lesions (CIN2+) by performing a preliminary evaluation of a new HPV test. Cervical specimens were obtained from 200 women referred for a colposcopy. Samples were tested using both Anyplex™ II HR-HPV as well as OncoPredict HPV^® Screening (SCR) and quantitative typing (QT). Using a cycle threshold cutoff (Ct) of 36.8 for the SCR assay and 1.27 log₁₀ (viral copies/10⁴ cells) for the QT assay, relative clinical sensitivity for CIN2+ and relative clinical specificity for CIN2− as compared to Anyplex™ II HR-HPV were, respectively, 0.92 and 1.00 for SCR and 1.35 and 1.24 for QT. The distribution of high-risk HPV (HR-HPV) genotypes (p = 0.009) as well as the viral copy numbers (CIN2−: 3.7 log₁₀ (viral copies/10⁴ human cells); CIN2+: 4.3 log₁₀ (viral copies/10⁴ human cells); p = 0.047) were found to differ in women with high- and low-grade cervical lesions, suggesting a possible role of HPV genotyping and normalized viral load as potential biomarkers to identify women at increased risk of cervical lesions. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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9 pages, 1643 KiB

Open AccessCommunication

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species

by Massimiliano Bergallo, Vivian Tullio, Janira Roana, Valeria Allizond, Narcisa Mandras, Valentina Daprà, Maddalena Dini, Sara Comini, Lorenza Cavallo, Stefano Gambarino, Anna Maria Cuffini and Giuliana Banche

Int. J. Mol. Sci. 2022, 23(23), 15066; https://doi.org/10.3390/ijms232315066 - 1 Dec 2022

Cited by 3 | Viewed by 2155

Abstract

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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18 pages, 3516 KiB

Open AccessArticle

Development and Clinical Validation of RT-LAMP-Based Lateral-Flow Devices and Electrochemical Sensor for Detecting Multigene Targets in SARS-CoV-2

by Apoorva Saxena, Pawankumar Rai, Srishti Mehrotra, Samiya Baby, Suman Singh, Vikas Srivastava, Smriti Priya and Sandeep K. Sharma

Int. J. Mol. Sci. 2022, 23(21), 13105; https://doi.org/10.3390/ijms232113105 - 28 Oct 2022

Cited by 9 | Viewed by 2516

Abstract

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL⁻¹ for N, ORF1ab, E genes and 100 agµL⁻¹ for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL⁻¹ for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL⁻¹ and log 0.067 µA/pg µL⁻¹/mm², respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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8 pages, 1345 KiB

Open AccessBrief Report

Novel Divergent Members of the Kitrinoviricota Discovered through Metagenomics in the Intestinal Contents of Red-Backed Voles (Clethrionomys gapperi)

by Marta Canuti, Bruce Rodrigues, Andrew S. Lang, Suzanne C. Dufour and Joost T. P. Verhoeven

Int. J. Mol. Sci. 2023, 24(1), 131; https://doi.org/10.3390/ijms24010131 - 21 Dec 2022

Cited by 1 | Viewed by 1478

Abstract

Metagenomic methods are powerful tools to investigate viral diversity in biological or environmental samples and to identify previously unknown viruses. We used RNA metagenomics to identify, in the gut of red-backed voles, the nearly complete genomes of two novel members of the Kitrinoviricota, a phylum including viruses with positive-sense ssRNA genomes encoding an RNA-directed RNA polymerase. The genome of a novel member of the Tombusviridae presented four open reading frames (ORFs); a −1 frameshift is potentially involved in generating the viral replicase. This sequence was part of a phylogenetic clade that did not include any officially classified species. The second genome presented a large ORF coding for a viral polyprotein containing the typical protein domains common to flexiviruses. The sequence clustered with currently known members of the Deltaflexiviridae. Both viruses appear to represent the first members of novel species in yet undefined genera. The identified viruses likely originated from the vole diet as members of the two viral families are known to infect plants and fungi, respectively. Investigating public databases demonstrated that a much higher richness than currently recognized exists for these two viral families, highlighting the need to update taxonomy systems and possibly also include genomes identified through metagenomics. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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8 pages, 1379 KiB

Open AccessBrief Report

Newly Designed Primers for the Sequencing of the inlA Gene of Lineage I and II Listeria monocytogenes Isolates

by Giulia Magagna, Guido Finazzi and Virginia Filipello

Int. J. Mol. Sci. 2022, 23(22), 14106; https://doi.org/10.3390/ijms232214106 - 15 Nov 2022

Cited by 2 | Viewed by 1391

Abstract

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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9 pages, 693 KiB

Open AccessBrief Report

Identification and Molecular Characterization of a Divergent Asian-like Canine Parvovirus Type 2b (CPV-2b) Strain in Southern Italy

by Giorgia Schirò, Francesco Mira, Marta Canuti, Stefano Vullo, Giuseppa Purpari, Gabriele Chiaramonte, Santina Di Bella, Vincenza Cannella, Vincenzo Randazzo, Calogero Castronovo, Domenico Vicari and Annalisa Guercio

Int. J. Mol. Sci. 2022, 23(19), 11240; https://doi.org/10.3390/ijms231911240 - 24 Sep 2022

Cited by 5 | Viewed by 1725

Abstract

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains. Full article

(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)

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