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Pathogens
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Molecular Diagnostic and Epidemiology of Viral Infections
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Molecular Diagnostic and Epidemiology of Viral Infections

A special issue of Pathogens (ISSN 2076-0817). This special issue belongs to the section "Viral Pathogens".

Deadline for manuscript submissions: closed (30 September 2022) | Viewed by 21263

Special Issue Editors

Dr. Dan Oţelea

E-Mail Website
Guest Editor
Molecular Diagnostics Laboratory, National Institute for Infectious Diseases, Bucharest, Romania
Interests: virology; HIV; molecular diagnostics and epidemiology
Dr. Snjezana Zidovec Lepej

E-Mail Website
Guest Editor
Department of Immunological and Molecular Diagnostics, University Hospital for Infectious Diseases, Zagreb, Croatia
Interests: molecular diagnostics; virology; immunology; HIV; viral hepatitis; HPV; SARS-CoV-2
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The necessity for rapid detection and characterization of SARS-CoV-2 pushed molecular diagnostics and epidemiology right to the forefront of medical attention. Molecular diagnostic tools have steadily evolved over the last two decades, but the onset of the COVID-19 pandemic has dramatically accelerated the pace of development within the field. Laboratory infrastructure and expertise in molecular diagnostics have expanded to unprecedented levels. Of note, at the beginning of April 2021, the GISAID database listed more than 993,000 SARS-CoV-2 full genome sequences. As a comparison, the number of HIV-1 (a major focus for several years) entries in the GenBank database was 960,000.

Has this expansion of molecular data modified the way we approach pathogen detection? Will our approach to other pathogens be influenced (and how) by these recent developments? PCR has already been a mainstay in approaching the patient with infectious pathology; will sequencing enter the standard clinical practice as well? Furthermore, can lessons learned from the COVID-19 experience be extrapolated to other pathologies? Are we better prepared for future challenges? We kindly invite you to share with us data that might shed light on these topics.

Dr. Dan Oţelea
Dr. Snjezana Zidovec Lepej
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pathogens is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • molecular diagnostics
  • molecular epidemiology
  • viral diseases

Published Papers (9 papers)

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Research

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12 pages, 1207 KiB  
Article
A Portable Diagnostic Assay, Genetic Diversity, and Isolation of Seoul Virus from Rattus norvegicus Collected in Gangwon Province, Republic of Korea
by Kyungmin Park, Seung-Ho Lee, Jongwoo Kim, Jingyeong Lee, Geum-Young Lee, Seungchan Cho, Juyoung Noh, Jeewan Choi, Juwon Park, Dong-Hyun Song, Se Hun Gu, Hyeongseok Yun, Jung-Eun Kim, Daesang Lee, Il-Ung Hwang, Won-Keun Kim and Jin-Won Song
Pathogens 2022, 11(9), 1047; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11091047 - 14 Sep 2022
Cited by 2 | Viewed by 2200
Abstract
Seoul virus (SEOV), an etiological agent for hemorrhagic fever with renal syndrome, poses a significant public health threat worldwide. This study evaluated the feasibility of a mobile Biomeme platform for facilitating rapid decision making of SEOV infection. A total of 27 Rattus norvegicus [...] Read more.
Seoul virus (SEOV), an etiological agent for hemorrhagic fever with renal syndrome, poses a significant public health threat worldwide. This study evaluated the feasibility of a mobile Biomeme platform for facilitating rapid decision making of SEOV infection. A total of 27 Rattus norvegicus were collected from Seoul Metropolitan City and Gangwon Province in Republic of Korea (ROK), during 2016–2020. The serological and molecular prevalence of SEOV was 5/27 (18.5%) and 2/27 (7.4%), respectively. SEOV RNA was detected in multiple tissues of rodents using the Biomeme device, with differences in Ct values ranging from 0.6 to 2.1 cycles compared to a laboratory benchtop system. Using amplicon-based next-generation sequencing, whole-genome sequences of SEOV were acquired from lung tissues of Rn18-1 and Rn19-5 collected in Gangwon Province. Phylogenetic analysis showed a phylogeographical diversity of rat-borne orthohantavirus collected in Gangwon Province. We report a novel isolate of SEOV Rn19-5 from Gangwon Province. Our findings demonstrated that the Biomeme system can be applied for the molecular diagnosis of SEOV comparably to the laboratory-based platform. Whole-genome sequencing of SEOV revealed the phylogeographical diversity of orthohantavirus in the ROK. This study provides important insights into the field-deployable diagnostic assays and genetic diversity of orthohantaviruses for the rapid response to hantaviral outbreaks in the ROK. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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11 pages, 1340 KiB  
Article
Epidemic Characteristics of HIV Drug Resistance in Hefei, Anhui Province
by Shan Zheng, Jianjun Wu, Jingjing Hao, Dong Wang, Zhongwang Hu, Lei Liu, Chang Song, Jing Hu, Yanhua Lei, Hai Wang, Lingjie Liao, Yi Feng, Yiming Shao, Yuhua Ruan and Hui Xing
Pathogens 2022, 11(8), 866; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11080866 - 31 Jul 2022
Cited by 4 | Viewed by 1334
Abstract
To study the characteristics of HIV pretreatment drug resistance (PDR) and acquired drug resistance (ADR) in Hefei, a cross-sectional survey was used to collect 816 samples from newly reported HIV infections from 2017 to 2020 and 127 samples from HIV infections with virological [...] Read more.
To study the characteristics of HIV pretreatment drug resistance (PDR) and acquired drug resistance (ADR) in Hefei, a cross-sectional survey was used to collect 816 samples from newly reported HIV infections from 2017 to 2020 and 127 samples from HIV infections with virological failure from 2018 to 2019 in Hefei. HIV drug resistance levels and drug resistance mutations were interpreted using the Stanford Drug Resistance Database. Molecular networks were constructed by HIV-TRACE. Among the newly reported infections in Hefei, the prevalence of PDR was 6.4% (52/816). The drug resistance mutations were mainly V179E/D/T (12.4%), K103N (1.3%), and V106I/M (1.3%). In addition, it was found that the CRF55_01B subtype had a higher drug resistance rate than other subtypes (p < 0.05). Molecular network analysis found that K103N and V179E may be transmitted in the cluster of the CRF55_01B subtype. The prevalence of ADR among HIV infections with virological failure was 38.6% (49/127), and the drug resistance mutations were mainly M184V (24.4%), K103N/S (15.7%), Y181C (11.0%), G190S/A/E (10.2%), and V106M/I (10.2%). The molecular network was constructed by combining HIV infections with virological failure and newly reported infections; M184V and Y181C may be transmitted between them. The chi-square trend test results indicated that the higher the viral load level, the greater the number of newly reported infections linked to the infections with virological failure in the molecular network. In conclusion, interventions should focus on infections of the CRF55_01B subtype to reduce the transmission of drug-resistant strains. However, improving the treatment effect of HIV infections is beneficial for reducing the second-generation transmission of HIV. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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23 pages, 1784 KiB  
Article
Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia
by Petra Simicic, Anamarija Slovic, Leona Radmanic, Adriana Vince and Snjezana Zidovec Lepej
Pathogens 2022, 11(7), 808; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11070808 - 19 Jul 2022
Viewed by 1517
Abstract
Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region [...] Read more.
Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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8 pages, 707 KiB  
Communication
SARS-CoV-2 Omicron Variant in Croatia—Rapid Detection of the First Case and Cross-Border Spread
by Ivana Ferenčak, Mihaela Obrovac, Ljiljana Žmak, Josipa Kuzle, Goranka Petrović, Tatjana Vilibić-Čavlek, Dragan Jurić, Anita Jurić, Željka Hruškar, Krunoslav Capak, Vladimir Stevanović, Maja Milanović, Marija Govedarica, Danijela Vujošević and Irena Tabain
Pathogens 2022, 11(5), 511; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11050511 - 26 Apr 2022
Cited by 4 | Viewed by 1937
Abstract
Background: Due to rapid spread, the Omicron variant has become the dominant SARS-CoV-2 variant responsible for infections worldwide. We present the first detection of the Omicron variant in Croatia which resulted in rapid cross-border spreading. Methods: Whole-genome sequencing was performed using the Illumina [...] Read more.
Background: Due to rapid spread, the Omicron variant has become the dominant SARS-CoV-2 variant responsible for infections worldwide. We present the first detection of the Omicron variant in Croatia which resulted in rapid cross-border spreading. Methods: Whole-genome sequencing was performed using the Illumina MiniSeq sequencing system. SARS-CoV-2 lineages were identified using the PANGOLIN and GISAID databases. Results: The first case of the Omicron variant (BA.1.17) emerged in Croatia after a workshop held in Zagreb in November 2021. The patient reported a history of previous COVID-19 and received two doses of an mRNA vaccine. Three additional cases were detected among Croatian participants of the workshop. At the beginning of December, SARS-CoV-2 infection was confirmed in one participant from Montenegro and her husband. Phylogenetic analysis showed that the detected Omicron variants were closely related to the first Croatian case, confirming the connection with the workshop outbreak and rapid cross-border spreading. Subsequent analyses of SARS-CoV-2 positive samples in Croatia showed the rapid introduction of the Omicron variant and depletion of the Delta variant resulting in the fifth pandemic wave. Conclusions: Genomic monitoring and early detection of novel SARS-CoV-2 variants are essential to implement timely epidemiological interventions and reduce further transmission in the population. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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14 pages, 1637 KiB  
Article
Comparative Evaluation of Six SARS-CoV-2 Real-Time RT-PCR Diagnostic Approaches Shows Substantial Genomic Variant–Dependent Intra- and Inter-Test Variability, Poor Interchangeability of Cycle Threshold and Complementary Turn-Around Times
by Rok Kogoj, Misa Korva, Nataša Knap, Katarina Resman Rus, Patricija Pozvek, Tatjana Avšič-Županc and Mario Poljak
Pathogens 2022, 11(4), 462; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11040462 - 12 Apr 2022
Cited by 7 | Viewed by 2170
Abstract
Several professional societies advise against using real-time Reverse-Transcription PCR (rtRT-PCR) cycle threshold (Ct) values to guide clinical decisions. We comparatively assessed the variability of Ct values generated by six diagnostic approaches by testing serial dilutions of well-characterized isolates of 10 clinically most relevant [...] Read more.
Several professional societies advise against using real-time Reverse-Transcription PCR (rtRT-PCR) cycle threshold (Ct) values to guide clinical decisions. We comparatively assessed the variability of Ct values generated by six diagnostic approaches by testing serial dilutions of well-characterized isolates of 10 clinically most relevant SARS-CoV-2 genomic variants: Alpha, Beta, Gamma, Delta, Eta, Iota, Omicron, A.27, B.1.258.17, and B.1 with D614G mutation. Comparison of three fully automated rtRT-PCR analyzers and a reference manual rtRT-PCR assay using RNA isolated with three different nucleic acid isolation instruments showed substantial inter-variant intra-test and intra-variant inter-test variability. Ct value differences were dependent on both the rtRT-PCR platform and SARS-CoV-2 genomic variant. Differences ranging from 2.0 to 8.4 Ct values were observed when testing equal concentrations of different SARS-CoV-2 variants. Results confirm that Ct values are an unreliable surrogate for viral load and should not be used as a proxy of infectivity and transmissibility, especially when different rtRT-PCR assays are used in parallel and multiple SARS-CoV-2 variants are circulating. A detailed turn-around time (TAT) comparative assessment showed substantially different TATs, but parallel use of different diagnostic approaches was beneficial and complementary, allowing release of results for more than 81% of non-priority samples within 8 h after admission. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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12 pages, 699 KiB  
Article
Detection of the ORF1 Gene Is an Indicator of the Possible Isolation of Severe Acute Respiratory Syndrome Coronavirus 2
by Kazuya Shirato, Masatoshi Kakizaki, Yuriko Tomita, Miyuki Kawase and Makoto Takeda
Pathogens 2022, 11(3), 302; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11030302 - 27 Feb 2022
Cited by 7 | Viewed by 4440
Abstract
In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity [...] Read more.
In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity of the patient. Due to the unique replication system of the coronavirus, primer/probe sets targeted nucleocapsid (N) and spike (S) protein detect the abundantly synthesized subgenomic RNAs as well as the virus genome, possibly making the assay unsuitable for estimation of the infectivity of the specimen, although it has an advantage for the diagnostic tests. In this study, the primer/probe set targeting the open reading frame 1a (ORF1a) gene was developed to specifically detect viral genomic RNA. Then the relation between the ORF1a signal and infectivity of the clinical specimens was validated by virus isolation using VeroE6 cells, which constitutively express transmembrane protease, serine 2, (VeroE6/TMPRSS2). The analytical sensitivity of developed ORF1a set was similar to that of previously developed N and S sets. Nevertheless, in the assay of the clinical specimen, detection rate of the ORF1a gene was lower than that of the N and S genes. These data indicated that clinical specimens contain a significant amount of subgenomic RNAs. However, as expected, the isolation-succeeded specimen always showed an RT-PCR-positive signal for the ORF1a gene, suggesting ORF1a detection in combination with N and S sets could be a more rational indicator for the possible infectivity of the clinical specimens. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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11 pages, 1464 KiB  
Article
Epidemiology and Molecular Transmission Characteristics of HIV in the Capital City of Anhui Province in China
by Shan Zheng, Jianjun Wu, Zhongwang Hu, Mengze Gan, Lei Liu, Chang Song, Yanhua Lei, Hai Wang, Lingjie Liao, Yi Feng, Yiming Shao, Yuhua Ruan and Hui Xing
Pathogens 2021, 10(12), 1554; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10121554 - 29 Nov 2021
Cited by 5 | Viewed by 1772
Abstract
Hefei, Anhui province, is one of the cities in the Yangtze River Delta, where many people migrate to Jiangsu, Zhejiang and Shanghai. High migration also contributes to the HIV epidemic. This study explored the HIV prevalence in Hefei to provide a reference for [...] Read more.
Hefei, Anhui province, is one of the cities in the Yangtze River Delta, where many people migrate to Jiangsu, Zhejiang and Shanghai. High migration also contributes to the HIV epidemic. This study explored the HIV prevalence in Hefei to provide a reference for other provinces and assist in the prevention and control of HIV in China. A total of 816 newly reported people with HIV in Hefei from 2017 to 2020 were recruited as subjects. HIV subtypes were identified by a phylogenetic tree. The most prevalent subtypes were CRF07_BC (41.4%), CRF01_AE (38.1%) and CRF55_01B (6.3%). Molecular networks were inferred using HIV-TRACE. The largest and most active transmission cluster was CRF55_01B in Hefei’s network. A Chinese national database (50,798 sequences) was also subjected to molecular network analysis to study the relationship between patients in Hefei and other provinces. CRF55_01B and CRF07_BC-N had higher clustered and interprovincial transmission rates in the national molecular network. People with HIV in Hefei mainly transmitted the disease within the province. Finally, we displayed the epidemic trend of HIV in Hefei in recent years with the dynamic change of effective reproductive number (Re). The weighted overall Re increased rapidly from 2012 to 2015, with a peak value of 3.20 (95% BCI, 2.18–3.85). After 2015, Re began to decline and remained stable at around 1.80. In addition, the Re of CRF55_01B was calculated to be between 2.0 and 4.0 in 2018 and 2019. More attention needs to be paid to the rapid spread of CRF55_01B and CRF07_BC-N strains among people with HIV and the high Re in Hefei. These data provide necessary support to guide the targeted prevention and control of HIV. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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8 pages, 264 KiB  
Communication
The Predictive Value of Mutation Screening for Anticipating COVID-19 Waves
by Robert Hohan, Petre Milu, Simona Paraschiv, Corina Casangiu, Andreea Tudor, Ovidiu Vlaicu, Leontina Banica, Marius Surleac, Dragos Florea and Dan Otelea
Pathogens 2021, 10(11), 1464; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10111464 - 11 Nov 2021
Cited by 3 | Viewed by 1744
Abstract
Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and [...] Read more.
Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and associated deaths. Whole Genome Sequencing (WGS) continues to be the gold standard for molecular surveillance of the pandemics but other assays such as mutation genotyping can be used to reduce costs and allocated time. This study investigates the efficiency of mutation screening tests compared to WGS and their predictive value to anticipate future waves. A very high degree of fidelity for this type of assay was found, regardless of the method used. The positive predictive value (PPV) of 4/5 markers was over 95% for the detection of Alpha and Delta variants. By estimating the prevalence of the Alpha and Delta strains using genotyping assays and fitting the data to a mathematical model, a five week period between the point of exponential growth of variant prevalence and a drastic increase in case numbers was found. For that reason, raising awareness about the efficacy of mutation screening could help authorities adopt better measures in the future. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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Review

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11 pages, 1658 KiB  
Review
Progress in Prophylactic and Therapeutic EBV Vaccine Development Based on Molecular Characteristics of EBV Target Antigens
by Marija Rozman, Petra Korać, Karlo Jambrosic and Snjezana Židovec Lepej
Pathogens 2022, 11(8), 864; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11080864 - 30 Jul 2022
Cited by 12 | Viewed by 2829
Abstract
Epstein–Barr virus (EBV) was discovered in 1964 in the cell line of Burkitt lymphoma and became first known human oncogenic virus. EBV belongs to the Herpesviridae family, and is present worldwide as it infects 95% of people. Infection with EBV usually happens during [...] Read more.
Epstein–Barr virus (EBV) was discovered in 1964 in the cell line of Burkitt lymphoma and became first known human oncogenic virus. EBV belongs to the Herpesviridae family, and is present worldwide as it infects 95% of people. Infection with EBV usually happens during childhood when it remains asymptomatic; however, in adults, it can cause an acute infection known as infectious mononucleosis. In addition, EBV can cause wide range of tumors with origins in B lymphocytes, T lymphocytes, and NK cells. Its oncogenicity and wide distribution indicated the need for vaccine development. Research on mice and cultured cells as well as human clinical trials have been in progress for a few decades for both prophylactic and therapeutic EBV vaccines. The main targets of the vaccines are EBV envelope glycoproteins such as gp350 and EBV latent genes. The long wait for the EBV vaccine is due to the complexity of the EBV replication cycle and the wide range of its host cells. Although some strategies such as the use of dendritic cells and recombinant Vaccinia viral vectors have shown success, ongoing clinical trials using mRNA-based vaccines as well as new delivery systems as nanoparticles are yet to show the best choice of vaccine target and its production strategy. Full article
(This article belongs to the Special Issue Molecular Diagnostic and Epidemiology of Viral Infections)
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