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Article

Epidemiological, Clinical and Phylogenetic Characteristics of West Nile Virus in Bulgaria, 2024

by
Kim Ngoc
1,
Ivan Stoikov
1,
Ivelina Trifonova
2,
Elitsa Panayotova
1,
Iva Trifonova
1 and
Iva Christova
1,*
1
Department of Microbiology, National Center of Infectious and Parasitic Diseases, 1504 Sofia, Bulgaria
2
Department of Virology, National Center of Infectious and Parasitic Diseases, 1233 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(4), 82; https://doi.org/10.3390/microbiolres16040082
Submission received: 21 February 2025 / Revised: 31 March 2025 / Accepted: 2 April 2025 / Published: 4 April 2025
Browse Figure
Versions Notes

Abstract

:
West Nile Virus (WNV), a mosquito-borne pathogen, is a growing public health challenge across Europe. Environmental and anthropogenic factors have led to the spread of the virus to higher geographic latitudes, as well as to increased viral circulation and genetic diversity. Aims: This study aimed to describe the epidemiological, clinical, and laboratory characteristics of WNV cases in Bulgaria during 2024 and to investigate WNV phylogenetics. Epidemiological, clinical and laboratory data from 32 patients with confirmed or probable WNV infections were collected and analysed. Complete viral genomes were obtained from two samples using whole genome sequencing (WGS). Phylogenetic analysis was performed using the Nextstrain WNV analysis pipeline. Severe disease was observed in 21 patients, with three fatalities reported in older males with comorbidities. Phylogenetic analysis revealed that Bulgarian strains clustered within the Central/Southern European clade of lineage 2, closely related to Greek strains. Evidence suggested localised viral evolution following cross-border introduction from Greece. Our study provides a detailed clinical and laboratory characterization of the human WNV cases detected in Bulgaria in 2024. Improved diagnostic workflows, expanded laboratory resources and increased molecular surveillance are essential to better understand the burden of WNV infections in Bulgaria, as well as to follow the evolution and spread of the virus.

1. Introduction

West Nile Virus (WNV) is a mosquito-borne virus belonging to the Orthoflavivirus genus within the Flaviviridae family. Birds serve as the main reservoir for WNV while humans are accidental dead-end hosts. The virus is transmitted to humans primarily through the bites of infected Culex mosquitoes. Other modes of transmission are rare and include blood transfusion, organ transplantation, and maternal-foetal transmission [1,2,3].
Most WNV infections are asymptomatic, with approximately 80% of cases showing no apparent illness. The majority of symptomatic cases present with a self-limiting febrile illness characterised by fever, headache, fatigue, myalgia, and occasionally a rash. In rare cases, WNV can cause severe neuroinvasive disease, especially in older or immunocompromised individuals. Neurological involvement may manifest as encephalitis, meningitis, or acute flaccid paralysis, which could lead to long-term neurological sequelae or death [4,5,6].
Since its first identification, WNV has been a growing challenge for public health systems across Europe [7,8]. As with other vector-borne infections, WNV circulation is highly influenced by various ecological and anthropogenic factors, such as changes in atmospheric conditions, ecosystem structures and land use. The rise in global temperatures has been a major contributor to the spread of the virus to higher latitudes, as well as to the increase in new cases in endemic regions [7,8,9,10]. In this context, whole genome sequencing (WGS) and phylogenetic analysis have become invaluable for tracking viral evolution and transmission patterns, improving the monitoring and management of WNV outbreaks [11].
Several WNV lineages have been established, with lineages 1 and 2 most frequently associated with human disease. Initially, lineage 2 isolates were found predominantly in Sub-Saharan Africa [5]. However, since its first detection in Europe in 2004, the lineage has quickly spread across the region, causing several large outbreaks over the years [6,7,8,9]. In 2018, lineage 2 was primarily responsible for a significant peak in WNV infections in Europe, with the number of autochthonous infections exceeding the total number of cases from the preceding seven years [12]. Bulgaria experienced a considerable increase in human cases that year (n = 15), compared to the average of 1.67 cases annually over the preceding three years [13,14].
Since 2018, Bulgaria has reported an average of two human WNV cases per year [15]; however, a significant increase was observed in 2024 [16]. This study aims to investigate the epidemiological aspects and describe the clinical and laboratory characteristics of all human WNV infection cases in Bulgaria in 2024.

2. Materials and Methods

2.1. Human WNV Infection Cases

Thirty-two patients from Bulgaria were diagnosed with probable or confirmed cases of WNV infection between August 2024 and November 2024. A confirmed WNC case requires at least one of the following: the isolation of WNV or the detection of WNV nucleic acid in the blood or cerebrospinal fluid (CSF); a WNV-specific IgM antibody response in CSF; or the detection of high-titre WNV IgM and WNV IgG in serum, confirmed by neutralization. A probable WNV case is defined as any patient presenting with fever, encephalitis, or meningitis, with either an epidemiological link or a WNV-specific antibody response in serum [17]. Demographic, epidemiological, and clinical data for the patients were made available through epidemiological investigation forms provided by the regional health inspectorates. Serum samples were available for all patients, urine samples were collected in 12 cases, cerebrospinal fluid was collected in 6, and blood was collected in 3. Patient samples were tested at the National Reference Laboratory for Vector-borne infections, part of the National Center of Infectious and Parasitic Diseases (NCIPD), Sofia. The majority of samples were sent from hospitals across the country, while a smaller number were sent from private laboratories.

2.2. RT-qPCR and Serological Testing

RNA extraction was performed with a commercial kit (QIAamp Viral RNA, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
Reverse transcriptase (RT) real-time polymerase chain reaction (PCR) testing was performed with primers and probes based on the protocol described by Linke [18].
For serological testing, enzyme-linked immunosorbent assays (ELISA) were employed to detect WNV-specific Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies. Samples were tested using commercially available ELISA kits (Euroimmun, Lübeck, Germany) in accordance with the manufacturer’s instructions. All patients underwent both RT-qPCR and serological testing.

2.3. Whole Genome Sequencing

Target enrichment next-generation sequencing was performed using the Viral Surveillance Panel v2 on a MiSeq platform with the MiSeq V3 reagent kit (2 × 300 bp) (Illumina, San Diego, CA, USA). Sequencing was only attempted with RT-qPCR-positive samples with a cycle threshold (Ct) value below 30.

2.4. Bioinformatic Analysis

The quality of raw sequencing data was assessed using FastQC v0.12.1 [19]. The quality trimming and filtering of the raw reads were performed using fastp v0.23.4 [20]. Processed reads were mapped against a reference genome sequence (GenBank accession number: NC_001563.2), and consensus sequences were generated with default parameters using Geneious Prime® 2023.2.1 [21]. The resulting assemblies were annotated with Vigor4 [22].
A time-resolved phylogenetic tree was constructed using curated data and a phylogenetic analysis pipeline from the Nextstrain WNV GitHub repository (https://github.com/nextstrain/WNV (accessed on 25 December 2024)), which implements WNV-specific analysis for the Nextstrain platform [23]. We used the global WNV build with AF260968 as the root sequence [24]. Our isolates were integrated into the existing dataset, and non-European strains were filtered out to focus on regional phylogenetics. The tree was visualized in Auspice v2.59.1 [25].

2.5. Data Availability

The complete genome sequences of the two Bulgarian strains have been deposited in GenBank under the accession numbers PQ793759 (v112_24) and PQ793760 (v123_24).

3. Results

3.1. Demographics and Clinical Presentation

In 2024, a total of 32 cases of confirmed or probable WNV infection were identified among Bulgarian citizens. The patients had a mean age of 59 years, with 8 patients under 50, 13 patients aged between 50 and 69, and 11 aged 70 years or older. Among them, 20 (62.5%) were male and 12 (37.5%) were female. The most common symptoms at disease onset included fever (n = 25, 78.1%), headache (n = 16, 50%), gastrointestinal symptoms (n = 13, 40.6%), malaise/fatigue (n = 11, 34.4%), arthralgia and/or myalgia (n = 8, 25%), rash (n = 8, 25%), and chills (n = 7, 21.9%). Other reported manifestations included dyspnoea and ocular manifestations (Table 1).
Severe disease was observed in 21 patients (65.6%), all of whom had neurological involvement and presented with symptoms such as headache, disorientation, somnolence, neck stiffness, seizures, tremors, psychomotor agitation, muscle weakness or increased muscle tone in the limbs. Of these patients, 17 (81%) were male and 4 were female (19%), with a mean age of 61 years. Nine patients had at least one known underlying condition, including congestive heart failure, Parkinson’s disease, diabetes mellitus, arterial hypertension, chronic renal failure, chronic obstructive pulmonary disease, and alcohol abuse. Mild to moderate disease was recorded in ten (31.3%) patients; their mean age was 52 years, while seven (70%) were female and three (30%) were male (Table 1). No clinical data were available in one case.

3.2. Fatal Outcomes

Three fatalities (9.4%) were reported, all involving male patients from Sofia City province with underlying comorbidities. The first case was a 73-year-old man presenting with fever, chills, and lower limb weakness. Despite initial treatment with antibiotics, he rapidly deteriorated and was hospitalised, requiring mechanical ventilation and falling into a coma. WNV RNA was detected in the patient’s urine, blood, and serum, with serum IgM positivity confirmed. The second case was a 61-year-old man with Parkinson’s disease, admitted with acute kidney failure and signs of neuroinfection. He presented with high fever, limb weakness, malaise, dyspnoea, and oliguria/anuria. The patient became unresponsive, hemodynamically unstable, and died on the sixth day. Serum and CSF testing confirmed WNV IgM positivity. The third case was a 55-year-old man admitted to the emergency room after fainting. He had psychomotor agitation, neck stiffness, increased muscle tone in the lower limbs, and a contused lacerated wound in the occipital region. His condition required admission to an intensive care unit, where he was intubated and put on mechanical ventilation. The patient died on the 11th day. Diagnostics revealed multi-infarct leukoencephalopathy and brain atrophy on CT imaging, as well as IgM positivity for WNV in the serum and CSF. Pre-existing medical conditions included arterial hypertension, diabetes mellitus and chronic renal failure.

3.3. Laboratory Findings

In eight patients, diagnosis was confirmed by the detection of WNV RNA in the urine, blood, CSF and/or serum by RT-qPCR (Table 1). Four additional patients were classified as confirmed cases based on the detection of specific IgM and/or IgG antibodies in the CSF. The remaining cases were classified as probable based on positive serological findings alone. Serum IgM antibodies were present in all patients in this study. IgG antibodies were initially positive in 15 cases and borderline in 4. Two patients with borderline and three patients with negative serum IgG on initial testing had developed positive IgG antibodies upon follow-up testing. Samples for initial WNV testing were collected, on average, 15 days after disease onset (range: 1–62 days). In 19 cases, initial testing was conducted in external laboratories using only serological methods. Samples with positive IgM antibody results were subsequently referred to the National reference laboratory for confirmation by RT-qPCR and serological testing.

3.4. Epidemiological Data

The epidemiological data showed that 19 patients had not travelled outside the country in the preceding three weeks before disease onset. Eight patients became ill while visiting Greece or shortly after. In five cases, no travel data were available. Most patients (n = 23, 71.9%) resided in Sofia City province, with single cases reported in Haskovo, Kardzhali, Sliven, Varna, and Plovdiv.

3.5. Phylogenetic and Amino Acid Analysis

Complete genome sequences were obtained successfully for two strains from urine samples. The two samples were collected from patients with no history of travel outside the country. Phylogenetic analysis confirmed that the two sequences belonged to the Central/Southern European clade of lineage 2 (Figure 1). They clustered closely with Greek sequences from 2019 to 2022, forming a distinct sub-branch within the Greek branch. The whole branch itself was characterised by three amino acid substitutions: I520V in the non-structural protein 5 (NS5), T157A in the envelope glycoprotein (env), and S73P (relative to the parent node) in the membrane glycoprotein precursor (prM): (the latter two were previously described by Tsioka et al. [26]). The two Bulgarian sequences had an additional substitution (M223I) relative to the Greek sequences in the non-structural protein 2A (NS2A). This substitution was present in only two other sequences from lineage 2 (LR743422 and KP780840), but was prevalent among sequences from lineage 1. Furthermore, the two sequences from this study differed from one another due to an A38V substitution in the non-structural protein 3 (NS3). This substitution was seen only in two other sequences from lineage 2 (LR743434 and LR743437), as well as in sequences from lineage 3 and 4. The distribution of WNV sequences based on the amino acids at the NS2A 223 and NS3 38 positions is shown in Supplementary Figures S1 and S2. The complete phylogenetic tree is available in Supplementary Figure S3.

4. Discussion

In 2024, 32 human cases of WNV infections were recorded in Bulgaria, which represents a significant increase compared to prior years [15]. Similar to previous studies, the majority of severe cases occurred in older adults, and particularly in men, with underlying health conditions such as diabetes mellitus, cardiovascular, pulmonary, renal and neurodegenerative diseases contributing to severe outcomes [27,28,29]. Three fatalities were recorded among older male patients with pre-existing conditions. Consistent with previous Bulgarian cases and ECDC surveillance reports, neuroinvasive disease was predominant among severe cases, with the most common neurological symptoms including disorientation, neck stiffness, seizures, headache and tremors. Systemic complications like pneumonia and pancreatitis, also noted in earlier studies, remain relevant for managing WNV patients [30,31,32,33]. In milder cases, the most frequent symptoms included fever, headache, malaise, gastrointestinal symptoms, and rash, similar to previous reports on non-neuroinvasive WNV infections [34,35]. The identification of milder cases may reflect improved public and clinical awareness, particularly as some patients sought testing based on public health awareness campaigns.
Although the number of identified cases were significantly higher than previous years, underdiagnosis remains a concern. Delayed testing, with samples typically collected an average of 15 days after disease onset, may reduce the sensitivity of molecular diagnostics, as viremia may have subsided during this period [36,37]. Furthermore, a reliance on serologic testing alone in the first days after disease onset could hinder accurate and timely diagnosis. This approach risks delays in confirmation and also likely leads to an underestimation of cases, as IgM antibodies may not yet be detectable in some individuals [38,39,40]. In 19 cases, the patient samples were initially tested using serological methods only before being referred to the National reference laboratory where RT-qPCR tests were performed. In one case, the patient had no detectable antibodies on the 3rd and 9th day after disease onset, with her first positive serological result being on the 19th day; after this, the samples were sent for RT-qPCR testing for the first time. Delays in molecular confirmation may have contributed to reduced diagnostic sensitivity, the underreporting of cases, and postponed diagnosis. This highlights the need for improved diagnostic workflows, emphasising the inclusion of molecular diagnostics at the earliest possible stage.
The first confirmed human WNV infections in Bulgaria were described in 2015, and WNV circulation in the country has been established through several seroepidemiological studies among humans, horses and birds, as well as through molecular studies involving mosquitoes and human samples [41,42,43,44,45,46]. In this study, all locally acquired cases were reported between August and November 2024, which could be due to the unusually high temperatures in the country [47]. In previous years, the last WNV cases in Bulgaria were reported no later than mid-October [16]. This could be linked to the higher temperatures observed in 2024, which remained elevated even in October, probably prolonging mosquito activity and virus circulation [47]. Similar patterns can be seen across Europe, where higher temperatures and agricultural activities are driving the acceleration of viral dispersal and genetic diversity [8]. The number of cases in the region for 2024 is above the mean monthly case count for the past 10 years. In countries neighbouring Bulgaria, the number of WNV cases in 2024 has also shown a noticeable increase compared to previous years, exceeding the annual average for the period 2015–2023 [33,48]. Greece reported 436 cases in 2024, Romania reported 353, Serbia reported 202 and North Macedonia reported 23 [33]. In all these countries, these numbers are the highest for the last ten years, except in Serbia, where 415 cases were reported in 2018 [48]. However, the ECDC data for 2024 may still be incomplete, as the official annual report for the 2024 transmission season has not been published at the time of writing. The year 2024 also marks the largest recorded geographic spread of WNV in Europe, with 212 regions reporting locally acquired human cases of WNV infection, compared to 137 regions in 2023 and 173 regions in 2018 [33]. For Bulgaria, the majority of cases in 2024 occurred in Sofia City province, where most WNV cases in the country have been historically documented, likely due to higher clinical awareness and diagnostic capacities. Isolated cases were also reported in other provinces, including Kardzhali, where WNV infection was documented for the first time.
The phylogenetic analysis of the two WNV genome sequences performed in this study placed them in the Central/Southern European clade of lineage 2, clustering together with strains from Greece. Genetic differences between the current (from 2024) and previous (from 2018 and 2015) Bulgarian strains indicate that the strains from 2024 originated from a separate introduction event. Their placement within the Greek branch suggests cross-border viral movement from Greece to Bulgaria, which likely occurred in the past 5 years. The additional mutations (NS2A: M223I, NS3: A38V) observed in the Bulgarian sequences imply that the virus has undergone localised evolution after its introduction. NS2A plays a role in the viral replication and modulation of host immune responses, while NS3 contains a protease and a helicase domain and is essential for viral protein processing and viral replication [49,50,51,52]. The observed mutations in these regions could potentially influence these functions, although their specific effects remain unexplored. Both strains were obtained from patients who denied traveling outside the country, pointing to the local transmission of the virus. While severe neuroinvasive disease was observed in both patients, there is insufficient evidence enabling us to directly link these mutations to increased pathogenicity; patient-specific factors—such as advanced age and comorbid conditions—likely played a more significant role. Nevertheless, these findings underline the importance of molecular surveillance in tracking the evolution and spread of WNV, as localised adaptations could impact viral transmissibility and virulence.
One limitation of this study is the relatively small number of WNV genome sequences obtained, with only two samples being successfully sequenced. The limited sample size restricts our ability to draw definitive conclusions about the genetic diversity of WNV in Bulgaria. Another limitation is that viral isolation from clinical samples could not be performed and positive serological results could not be confirmed through neutralisation testing due to limited resources. Without these methods, reliable case confirmation and the further investigation of the viral and immunological characteristics of the infection become more challenging.
In conclusion, our study provides a detailed clinical and laboratory characterization of human WNV cases detected in Bulgaria in 2024. The phylogenetic analysis indicates cross-border viral transmission from Greece to Bulgaria, as well as localised evolution. Improved diagnostic workflows and expanded resources for confirmatory testing are essential to better assess the prevalence and impact of WNV infections in Bulgaria.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres16040082/s1, Figure S1. Distribution of West Nile Virus (WNV) sequences based on the amino acid at position 223 in the non-structural protein 2 (NS2). Sequences from lineage 2 containing Isoleucine at the NS2A 223 site are annotated; Figure S2. Distribution of WNV sequences based on the amino acid at position 38 in the non-structural protein 3 (NS3). Sequences from lineage 2 containing Valine at the NS3 38 site are annotated; Figure S3. Phylogenetic tree of all complete WNV sequences from Europe, coloured by country of origin. Branch labels indicate the WNV lineage.

Author Contributions

Conceptualization, I.C. and K.N.; methodology, I.C. and I.T. (Iva Trifonova); software, I.S. and K.N.; validation, E.P., I.T. (Ivelina Trifonova), I.S. and I.T. (Iva Trifonova); formal analysis, K.N. and I.S.; investigation, E.P. and I.T. (Ivelina Trifonova); resources, I.C., I.T. (Iva Trifonova) and E.P.; data curation, K.N. and I.S.; writing—original draft preparation, K.N.; writing—review and editing, K.N., I.S. and I.T. (Ivelina Trifonova); visualization, K.N. and I.S.; supervision, I.C. and I.T. (Iva Trifonova); project administration, I.C.; funding acquisition, I.C. and K.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Program “Research, Innovation and Digitalisation for Smart Transformation” (PRIDST) 2021–2027, BG16RFPR002-1.014 “Sustainable Development of Centers of Excellence and Centers of Competence, including specific infrastructures or their consortia from the National Roadmap for Research Infrastructure (NRRI)”.

Institutional Review Board Statement

Ethical approval for this study was obtained from the Institutional review board at NCIPD (approval number 6/23.11.2023).

Informed Consent Statement

Patient consent was waived because the study does not involve any information that could lead to the identification of individual participants, as approved by the Institutional Review Board at NCIPD (approval number 6/23.11.2023).

Data Availability Statement

Data is contained within the article or Supplementary Materials.

Acknowledgments

K.N. has received research funding from the Bulgarian Ministry of Education and Science under the National Program “Young Scientists and Postdoctoral Students–2”.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
WNVWest Nile virus
WGSWhole genome sequencing
CSFCerebrospinal fluid
PCRPolymerase chain reaction
ELISAEnzyme-linked immunosorbent assay
NCIPDNational centre of infectious and parasitic diseases
ECDCEuropean Centre for Disease Prevention and Control

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Microbiolres 16 00082 g001
Figure 1. Phylogenetic analysis of West Nile Virus (WNV) lineage 2. Phylogenetic tree of all analysed WNV lineage 2 sequences from Europe, coloured by country of origin. Below is a close-up view of the branch containing the Bulgarian sequences from this study (red labels). Branch labels indicate amino acid substitutions that define the corresponding branches (not all branch labels are shown).
Figure 1. Phylogenetic analysis of West Nile Virus (WNV) lineage 2. Phylogenetic tree of all analysed WNV lineage 2 sequences from Europe, coloured by country of origin. Below is a close-up view of the branch containing the Bulgarian sequences from this study (red labels). Branch labels indicate amino acid substitutions that define the corresponding branches (not all branch labels are shown).
Microbiolres 16 00082 g001
Table 1. Clinical and laboratory characteristics of WNV cases.
Table 1. Clinical and laboratory characteristics of WNV cases.
Age/SexSymptomsUnderlying ConditionsOutcomePositive WNV Laboratory FindingsCase Classification
Mild/Moderate
30/FFever, malaise, headache, rash, eye painHashimoto thyroiditis, vitiligoRecoveredSerum IgM and IgG positiveProbable
46/FMalaise, rash, eye swelling RecoveredSerum IgM and IgG positiveProbable
57/FFever, chills, headache, rash RecoveredCSF IgM positive
Serum IgM positive		Confirmed
63/FFever, rash RecoveredSerum IgM positiveProbable
55/FFever, headache RecoveredSerum IgM positiveProbable
71/MFever, vomiting, stomach pain, diarrhoea, diplopia, myalgiaDiabetes mellitusRecoveredRT-qPCR positive (urine)
Serum IgM and IgG positive		Confirmed
41/MFever, toxic–infectious syndrome RecoveredRT-qPCR positive (blood)
Serum IgM positive		Confirmed
34/FFever, headache, nausea, vomiting, arthralgia RecoveredSerum IgM and IgG positiveProbable
50/MFever, malaise, myalgiaCancerRecoveredSerum IgM positiveProbable
74/FFever, malaise, headache, dark skin spots, cervical lymphadenopathy RecoveredSerum IgM and IgG positiveProbable
75/FN/AN/AN/ASerum IgM positive
Serum IgG borderline		Probable
Severe
73/MFever, chills, fatigue, lower limb weakness, comaCongestive heart diseaseFatalRT-qPCR positive (urine)
Serum IgM positive		Confirmed
60/MFever, fatigue, dyspnoea, oliguria/anuria, increased muscle tone in limbs, deteriorated general condition, unresponsivenessParkinson diseaseFatalSerum IgM positive
Serum IgG borderline		Probable
74/MFever, dyspnoea, fatigue, numbness and muscle pain, somnolence, unconsciousness, vomiting RecoveredRT-qPCR positive (serum)
Serum IgM and IgG positive		Confirmed
61/MFever, chills, arthralgia, myalgia, disoriented, psychoemotionally agitated, neck stiffness, positive Kernig’s sign, headache, vomitingBronchial asthma, anaemiaRecoveredRT-qPCR positive (CSF, urine, serum)
Serum IgM and IgG positive		Confirmed
49/MFever, chills, rash, jaw stiffness, tongue biting, difficulty communicating, myoclonic contractions of the limbs, hypersensitivity, hydrophobia, quantitative changes in consciousness, headache RecoveredCSF IgM and IgG positive
Serum IgM and IgG positive		Confirmed
66/MFever, rash, disoriented, difficulty communicating, deteriorated general condition, headache, vomitingUrothelial papilloma, diabetes mellitus with complications, chronic renal failure, chronic obstructive pulmonary disease, Hashimoto thyroiditisRecoveredSerum IgM positiveProbable
59/MFever, chills, fatigue, myalgia, rash, increased muscle tone in limbs, quantitative changes in consciousness, deteriorated general condition, hypersensitivity, headache RecoveredRT-qPCR positive (urine)
Serum IgM and IgG positive		Confirmed
55/MPsychomotor agitation, neck stiffness, positive Kernig’s sign, increased muscle tone in limbsArterial hypertension, diabetes mellitus, chronic renal failureFatalCSF IgM positive
Serum IgM and IgG positive		Confirmed
73/MFever, nausea, vomiting, tremors, deteriorated general condition RecoveredRT-qPCR positive (urine)
Serum IgM and IgG positive		Confirmed
67/MFever, myalgia, disoriented, inadequate, difficult to communicate with, immobile, deteriorated general condition, headache, nauseaArterial hypertension, goutRecoveredSerum IgM and IgG positiveProbable
57/MChills, headache, vomiting, dizziness RecoveredCSF IgM positive
Serum IgM and IgG positive		Confirmed
67/FFever, fatigue, nausea, myalgia, arthralgia, headache, diplopia, dizziness, bradyphasia RecoveredSerum IgM and IgG positiveProbable
90/MFever, dyspnoea, deteriorated general condition, unresponsive, clonic seizuresChronic obstructive pulmonary disease, chronic congestive heart disease, arterial hypertensionRecoveredSerum IgM and IgG positiveProbable
80/FFever, chills, fatigue, cough, headache, vomiting, confusion, unresponsive, left gaze deviation, mild seizures with apnoeic pauses, neck stiffness, positive Kernig’s sign; weak somatic reflexesArterial hypertension, diabetes mellitusRecoveredSerum IgM and IgG positiveProbable
55/MDeteriorated general condition, inadequate, difficult verbal contact, tremor of the upper limbs, exsiccosis, facial asymmetry, absent bilateral patellar and Achilles reflexes, unresponsive to pain, coma, hyperglycaemia, pleural effusionsAlcohol abuse, diabetes mellitus, chronic pancreatitisRecoveredSerum IgM and IgG positiveProbable
26/MFever, syncope, seizures, tongue biting, vomiting RecoveredSerum IgM positiveProbable
47/FFever, partially disoriented, bradypsychic, neck stiffness, severe headache, vomiting RecoveredSerum IgM and IgG positiveProbable
71/MFever, positive pathological reflexes, somnolence, severe headache, vomiting RecoveredRT-qPCR positive (urine)
Serum IgM and IgG positive		Confirmed
71/MBrain edema RecoveredSerum IgM positiveProbable
72/MFever, fatigue, myalgia, arthralgia, rash, headache, disorientation, dizziness, sleepiness; deteriorated general condition, unconsciousness RecoveredSerum IgM and IgG positiveProbable
17/FMeningoencephalitis RecoveredSerum IgM positiveProbable
N/A = not available, CSF = cerebrospinal fluid, RT-qPCR = quantitative reverse transcriptase polymerase chain reaction.
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Ngoc, K.; Stoikov, I.; Trifonova, I.; Panayotova, E.; Trifonova, I.; Christova, I. Epidemiological, Clinical and Phylogenetic Characteristics of West Nile Virus in Bulgaria, 2024. Microbiol. Res. 2025, 16, 82. https://doi.org/10.3390/microbiolres16040082

AMA Style

Ngoc K, Stoikov I, Trifonova I, Panayotova E, Trifonova I, Christova I. Epidemiological, Clinical and Phylogenetic Characteristics of West Nile Virus in Bulgaria, 2024. Microbiology Research. 2025; 16(4):82. https://doi.org/10.3390/microbiolres16040082

Chicago/Turabian Style

Ngoc, Kim, Ivan Stoikov, Ivelina Trifonova, Elitsa Panayotova, Iva Trifonova, and Iva Christova. 2025. "Epidemiological, Clinical and Phylogenetic Characteristics of West Nile Virus in Bulgaria, 2024" Microbiology Research 16, no. 4: 82. https://doi.org/10.3390/microbiolres16040082

APA Style

Ngoc, K., Stoikov, I., Trifonova, I., Panayotova, E., Trifonova, I., & Christova, I. (2025). Epidemiological, Clinical and Phylogenetic Characteristics of West Nile Virus in Bulgaria, 2024. Microbiology Research, 16(4), 82. https://doi.org/10.3390/microbiolres16040082

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