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10 pages, 445 KiB

Open AccessArticle

Detection of Chikungunya Virus RNA in Oral Fluid and Urine: An Alternative Approach to Diagnosis?

by Leile Camila Jacob-Nascimento, Moyra M. Portilho, Rosângela O. Anjos, Patrícia S. S. Moreira, Christine Stauber, Scott C. Weaver, Uriel Kitron, Mitermayer G. Reis and Guilherme S. Ribeiro

Viruses 2024, 16(2), 235; https://doi.org/10.3390/v16020235 - 02 Feb 2024

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Abstract

To evaluate whether oral fluids (OF) and urine can serve as alternative, non-invasive samples to diagnose chikungunya virus (CHIKV) infection via RT-qPCR, we employed the same RNA extraction and RT-qPCR protocols on paired serum, OF and urine samples collected from 51 patients with [...] Read more.

To evaluate whether oral fluids (OF) and urine can serve as alternative, non-invasive samples to diagnose chikungunya virus (CHIKV) infection via RT-qPCR, we employed the same RNA extraction and RT-qPCR protocols on paired serum, OF and urine samples collected from 51 patients with chikungunya during the acute phase of the illness. Chikungunya patients were confirmed through RT-qPCR in acute-phase sera (N = 19), IgM seroconversion between acute- and convalescent-phase sera (N = 12), or IgM detection in acute-phase sera (N = 20). The controls included paired serum, OF and urine samples from patients with non-arbovirus acute febrile illness (N = 28) and RT-PCR-confirmed dengue (N = 16). Nine (47%) of the patients with positive RT-qPCR for CHIKV in sera and two (17%) of those with CHIKV infection confirmed solely via IgM seroconversion had OF positive for CHIKV in RT-qPCR. One (5%) patient with CHIKV infection confirmed via serum RT-qPCR was positive in the RT-qPCR performed on urine. None of the negative control group samples were positive. Although OF may serve as an alternative sample for diagnosing acute chikungunya in specific settings, a negative result cannot rule out an infection. Further research is needed to investigate whether OF and urine collected later in the disease course when serum becomes RT-qPCR-negative may be helpful in CHIKV diagnosis and surveillance, as well as to determine whether urine and OF pose any risk of CHIKV transmission. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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18 pages, 872 KiB

Open AccessArticle

Diagnosis of Imported Dengue and Zika Virus Infections in Italy from November 2015 to November 2022: Laboratory Surveillance Data from a National Reference Laboratory

by Christina Merakou, Antonello Amendola, Claudia Fortuna, Giulia Marsili, Cristiano Fiorentini, Claudio Argentini, Eleonora Benedetti, Gianni Rezza, Francesco Maraglino, Martina Del Manso, Antonino Bella, Patrizio Pezzotti, Flavia Riccardo, Anna Teresa Palamara, Giulietta Venturi and The Arbovirus Working Group

Viruses 2024, 16(1), 50; https://doi.org/10.3390/v16010050 - 28 Dec 2023

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Abstract

Dengue (DENV) and Zika (ZIKV) viruses are mosquito-borne human pathogens. In Italy, the presence of the competent vector Aedes albopictus increases the risk of autochthonous transmission, and a national plan for arboviruses prevention, surveillance, and response (PNA 2020–2025) is in place. The results [...] Read more.

Dengue (DENV) and Zika (ZIKV) viruses are mosquito-borne human pathogens. In Italy, the presence of the competent vector Aedes albopictus increases the risk of autochthonous transmission, and a national plan for arboviruses prevention, surveillance, and response (PNA 2020–2025) is in place. The results of laboratory diagnosis of both viruses by the National Reference Laboratory for arboviruses (NRLA) from November 2015 to November 2022 are presented. Samples from 655 suspected cases were tested by both molecular and serological assays. Virus and antibody kinetics, cross-reactivity, and diagnostic performance of IgM ELISA systems were analysed. Of 524 cases tested for DENV, 146 were classified as confirmed, 7 as probable, while 371 were excluded. Of 619 cases tested for ZIKV, 44 were classified as confirmed, while 492 were excluded. All cases were imported. Overall, 75.3% (110/146) of DENV and 50% (22/44) of ZIKV cases were confirmed through direct virus detection methods. High percentages of cross reactivity were observed between the two viruses. The median lag time from symptoms onset to sample collection was 7 days for both DENV molecular (range 0–20) and NS1 ELISA (range 0–48) tests, with high percentages of positivity also after 7 days (39% and 67%, respectively). For ZIKV, the median lag time was 5 days (range 0–22), with 16% positivity after 7 days. Diagnostic performance was assessed with negative predictive values ranging from 92% to 95% for the anti-DENV systems, and of 97% for the ZIKV one. Lower positive predictive values were seen in the tested population (DENV: 55% to 91%, ZIKV: 50%). DENV and ZIKV diagnosis by molecular test is the gold standard, but sample collection time is a limitation. Serological tests, including Plaque Reduction Neutralization Test, are thus necessary. Co-circulation and cross-reactivity between the two viruses increase diagnostic difficulty. Continuous evaluation of diagnostic strategies is essential to improve laboratory testing. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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12 pages, 2184 KiB

Open AccessEditor’s ChoiceArticle

Nanoluciferase Reporter Zika Viruses as Tools for Assessing Infection Kinetics and Antibody Potency

by Yanqun Xu, Devin Vertrees, Yong He, Sanaz Momben-Abolfath, Xiaohong Li, Yambasu A. Brewah, Dorothy E. Scott, Krishnamurthy Konduru, Maria Rios and Evi B. Struble

Viruses 2023, 15(11), 2190; https://doi.org/10.3390/v15112190 - 31 Oct 2023

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Abstract

Zika virus (ZIKV) has become endemic in multiple tropical and subtropical regions and has the potential to become widespread in countries with limited prior exposure to this infection. One of the most concerning sequelae of ZIKV infection is the teratogenic effect on the [...] Read more.

Zika virus (ZIKV) has become endemic in multiple tropical and subtropical regions and has the potential to become widespread in countries with limited prior exposure to this infection. One of the most concerning sequelae of ZIKV infection is the teratogenic effect on the developing fetus, with the mechanisms of viral spread to and across the placenta remaining largely unknown. Although vaccine trials and prophylactic or therapeutic treatments are being studied, there are no approved treatments or vaccines for ZIKV. Appropriate tests, including potency and in vivo assays to assess the safety and efficacy of these modalities, can greatly aid both the research of the pathophysiology of the infection and the development of anti-ZIKV therapeutics. Building on previous work, we tested reporter ZIKV variants that express nanoluciferase in cell culture and in vivo assays. We found that these variants can propagate in cells shown to be susceptible to the widely used clinical isolate PRVABC59, including Vero and human placenta cell lines. When used in neutralization assays with bioluminescence as readout, these variants gave rise to neutralization curves similar to those produced by PRVABC59, while being better suited for performing high-throughput assays. In addition, the engineered reporter variants can be useful research tools when used in other in vitro and in vivo assays, as we illustrated in transcytosis experiments and a pilot study in guinea pigs. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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19 pages, 3450 KiB

Open AccessArticle

Serological and Molecular Epidemiology of Chikungunya Virus Infection in Vietnam, 2017–2019

by Thanh Vu Nguyen, Mya Myat Ngwe Tun, Minh Thang Cao, Huy Manh Dao, Chan Quang Luong, Thi Kim Loan Huynh, Thi Thanh Thuong Nguyen, Thi Nhu Dao Hoang, Kouichi Morita, Thi Quynh Mai Le, Quang Duy Pham, Yuki Takamatsu and Futoshi Hasebe

Viruses 2023, 15(10), 2065; https://doi.org/10.3390/v15102065 - 08 Oct 2023

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Abstract

Chikungunya fever is an acute febrile illness caused by the chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Since 1965, only a few studies with limited scope have been conducted on CHIKV in Vietnam. Thus, this study aimed to determine the seroprevalence [...] Read more.

Chikungunya fever is an acute febrile illness caused by the chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Since 1965, only a few studies with limited scope have been conducted on CHIKV in Vietnam. Thus, this study aimed to determine the seroprevalence and molecular epidemiology of CHIKV infection among febrile patients in Vietnam from 2017 to 2019. A total of 1063 serum samples from 31 provinces were collected and tested for anti-CHIKV IgM and IgG ELISA. The 50% focus reduction neutralization test (FRNT₅₀) was used to confirm CHIKV-neutralizing antibodies. Quantitative real-time RT–PCR (RT–qPCR) was performed to confirm the presence of the CHIKV genome. The results showed that 15.9% (169/1063) of the patients had anti-CHIKV IgM antibodies, 20.1% (214/1063) had anti-CHIKV IgG antibodies, 10.4% (111/1063) had CHIKV-neutralizing antibodies, and 27.7% (130/469) of the samples were positive in RT–qPCR analysis. The E1 CHIKV genome sequences were detected among the positive RT–qPCR samples. Our identified sequences belonged to the East/Central/South/African (ECSA) genotype, which has been prevalent in Vietnam previously, suggesting CHIKV has been maintained and is endemic in Vietnam. This study demonstrates a high prevalence of CHIKV infection in Vietnam and calls for an annual surveillance program to understand its impact. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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11 pages, 1086 KiB

Open AccessArticle

Burden of Chikungunya Virus Infection during an Outbreak in Myanmar

by Mya Myat Ngwe Tun, Aung Kyaw Kyaw, Khine Mya Nwe, Su Su Myaing, Ye Thu Win, Shingo Inoue, Yuki Takamatsu, Takeshi Urano, Hlaing Myat Thu, Saw Wutt Hmone, Kyaw Zin Thant and Kouichi Morita

Viruses 2023, 15(8), 1734; https://doi.org/10.3390/v15081734 - 14 Aug 2023

Cited by 1 | Viewed by 1165

Abstract

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central [...] Read more.

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central South African genotype of CHIKV occurred in Myanmar, and during this period, a cross-sectional study was conducted in two regions, Mandalay and Yangon, to examine the molecular and seropositivity rate of the CHIKV infection. The participants (1124) included dengue-suspected pediatric patients, blood donors, and healthy volunteers, who were assessed using molecular assays (quantitative real-time RT-PCR), serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assays), and neutralization tests. The tests confirmed the following positivity rates: 11.3% (127/1124) for the molecular assay, 12.4% (139/1124) for the anti-CHIKV IgM Ab, 44.5% (500/1124) for the anti-CHIKV IgG Ab, and 46.3% (520/1124) for the CHIKV neutralizing Ab. The highest rate for the molecular test occurred with the dengue-suspected pediatric patients. The seroprevalence rate through natural infection was higher in the healthy volunteers and blood donors than that in the pediatric patients. The results of this study will help stakeholders determine the criteria for choosing appropriate recipients when a CHIKV vaccine is introduced in Myanmar. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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15 pages, 294 KiB

Open AccessArticle

Novel Amplicon-Based Sequencing Approach to West Nile Virus

by Moussa Moïse Diagne, Marie Henriette Dior Ndione, Giulia Mencattelli, Amadou Diallo, El hadji Ndiaye, Marco Di Domenico, Diawo Diallo, Mouhamed Kane, Valentina Curini, Ndeye Marieme Top, Maurilia Marcacci, Maïmouna Mbanne, Massimo Ancora, Barbara Secondini, Valeria Di Lollo, Liana Teodori, Alessandra Leone, Ilaria Puglia, Alioune Gaye, Amadou Alpha Sall, Cheikh Loucoubar, Roberto Rosà, Mawlouth Diallo, Federica Monaco, Ousmane Faye, Cesare Cammà, Annapaola Rizzoli, Giovanni Savini and Oumar Faye Show full author list Hide full author list

Viruses 2023, 15(6), 1261; https://doi.org/10.3390/v15061261 - 27 May 2023

Cited by 1 | Viewed by 1760

Abstract

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements [...] Read more.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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7 pages, 474 KiB

Open AccessBrief Report

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study

by Antonio Mori, Andrea Matucci, Elena Pomari, Silvia Accordini, Chiara Piubelli, Annalisa Donini, Lavinia Nicolini and Concetta Castilletti

Viruses 2024, 16(1), 98; https://doi.org/10.3390/v16010098 - 08 Jan 2024

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Abstract

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical [...] Read more.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID₅₀ vs. 1 × 10⁴ TCID₅₀ limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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8 pages, 764 KiB

Open AccessBrief Report

Development of a Real-Time PCR Assay for the Detection of the Phlebovirus Fermo Virus

by Mattia Calzolari, Simone Russo, Katia Marzani, Gastone Dalmonte, Matteo Ricchi and Paolo Bonilauri

Viruses 2023, 15(10), 2082; https://doi.org/10.3390/v15102082 - 12 Oct 2023

Cited by 1 | Viewed by 807

Abstract

Fermo virus is a Phlebovirus that is increasingly reported in sand flies from northern Italy. The natural cycle is not fully understood, but the virus has been detected by direct methods only in sand flies. Although there is serological evidence that it can [...] Read more.

Fermo virus is a Phlebovirus that is increasingly reported in sand flies from northern Italy. The natural cycle is not fully understood, but the virus has been detected by direct methods only in sand flies. Although there is serological evidence that it can infect vertebrates, the virus has not been directly detected in animals or humans. Here, we have developed and reported a specific real-time PCR for Fermo virus. The availability of the described method will be useful to characterize the epidemiology of the FERV, ensuring, compared to previously available protocols, a more sensitive detection in insects and the possible detection in vertebrates to evaluate the presence of reservoirs and the pathogenic potential of the virus in humans or animals. Full article

(This article belongs to the Special Issue Arbovirus Diagnostics)

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