Cytokine profiles in Chikungunya virus-infected patients in Myanmar: a first report

Abstract

Chikungunya virus (CHIKV) is endemic in Myanmar and remains an important cause of arboviral febrile illness. However, cytokine responses during CHIKV infection in Myanmar remain poorly characterized. In this study, serum cytokine profiles were analyzed in CHIKV RT–qPCR-positive patients using the Human Cytokine Magnetic 10-Plex Panel. Seven cytokines were detectable, including IFN-γ, TNF-α, IL-4, IL-6, IL-8, IL-10, and GM-CSF. Compared with healthy controls, all detectable cytokines except TNF-α were significantly elevated in CHIKV-infected patients. Cytokine concentrations varied according to patient’s viral load and clinical presentation. These findings demonstrate distinct inflammatory and regulatory cytokine responses during CHIKV infection and provide insights into host immune responses in Myanmar CHIKV patients. Further studies are needed to clarify the cytokine responses associated with disease severity and viral burden.

Supplementary Information

The online version contains supplementary material available at 10.1186/s41182-026-01041-2.

Background

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that remains an important cause of arboviral febrile illness in endemic regions, including Myanmar [1]. CHIKV infection is associated with inflammatory immune responses characterized by cytokine production involved in antiviral defense and disease manifestations. Previous studies have reported altered cytokine expression during CHIKV infection, including elevated pro-inflammatory and regulatory cytokines associated with disease severity and viremia [1, 2 ]. Myanmar is a dengue-endemic setting, where CHIKV and DENV co-circulate, resulting in overlapping clinical presentations and occasional co-infections. Despite this epidemiological context, information on cytokine responses in CHIKV-infected patients from Myanmar remains limited. Characterizing these immune responses in an understudied CHIKV-endemic population provides important immunological data and enables comparison with findings reported from other endemic regions. We hypothesized that CHIKV infection is associated with altered circulating cytokine responses and that these immune responses vary according to virological and clinical characteristics. Therefore, this study analyzed serum cytokine profiles in CHIKV RT–qPCR-positive patients from Myanmar and explored cytokine concentrations according to viral load, disease severity, DENV co-infection and clinical presentation.

Methods

A total of 80 serum samples previously collected in Myanmar for arboviral studies [3, 4 ] were analyzed, including 72 CHIKV RT–qPCR-positive samples and 8 healthy controls. CHIKV mono-infection was defined as CHIKV infection without laboratory-confirmed DENV infection, whereas CHIKV–DENV co-infection was defined as concurrent laboratory confirmation of both viruses [5]. Clinical classification was based on the 2009 WHO dengue guidelines. Patients classified as dengue without warning signs (DWoWS) were categorized as non-severe CHIKV, whereas those classified as dengue with warning signs (DWWS) or severe dengue (SD) were categorized as severe CHIKV disease for exploratory analyses according to clinical presentation. Because the WHO dengue classification has not been validated specifically for CHIKV infection, these categories were used as a pragmatic clinical stratification and interpreted accordingly. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). CHIKV viral load was quantified by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) using TaqMan Fast Virus 1-Step Master Mix (Life Technologies, Carlsbad, CA, USA) and classified as low (< 10⁶ copies/mL) or high (≥ 10⁶ copies/mL). Serum cytokine concentrations were measured using the Human Cytokine Magnetic 10-Plex Panel (MilliporeSigma, Burlington, MA, USA) on the Luminex platform. Cytokines were grouped as pro-inflammatory (IFN-γ, IL-1β, IL-6, IL-8, TNF-α, GM-CSF), growth and activation-related (IL-2, IL-5), or regulatory/anti-inflammatory (IL-4, IL-10). Statistical analyses were performed using GraphPad Prism version 10. Cytokine levels were compared between two groups using Mann–Whitney U test, while correlations were assessed using Spearman correlation. P values < 0.05 were considered statistically significant. Because this study was exploratory, adjustments for multiple comparisons were not applied, and the results should be interpreted accordingly.

Results

Among the 10 cytokines included in the panel, seven (IFN-γ, TNF-α, IL-4, IL-6, IL-8, IL-10, and GM-CSF) were detected in the analyzed serum samples, whereas IL-1β, IL-2, and IL-5 were not detected. Compared with healthy controls, CHIKV-infected patients showed significantly elevated IFN-γ, IL-4, IL-6, IL-8/CXCL8, IL-10, and GM-CSF levels (Fig. 1).

Fig. 1.

CHIKV serum cytokine levels (log2 pg/mL) in CHIKV-infected patients (n = 72) and healthy controls (HC; n = 8). Cytokine concentrations are presented as mean ± SEM and were compared using Mann–Whitney U test. Exact p values are shown in the figure

CHIKV–DENV co-infected patients showed significantly higher IFN-γ and IL-10 levels than CHIKV mono-infected patients (Fig. 2).

Fig. 2.

Comparison of CHIKV serum cytokine levels (log2 pg/mL) between CHIKV mono-infected (n = 51) and CHIKV–DENV co-infected patients (n = 21). Cytokine concentrations are presented as mean ± SEM and were compared using Mann–Whitney U test. Exact p values are shown in the figure

Viremia analysis showed significantly higher TNF-α and IL-8 levels in patients with high CHIKV viral load, whereas IFN-γ and IL-10 were significantly higher in patients with lower viral load (Fig. 3).

Fig. 3.

Serum cytokine levels according to CHIKV viremia: low viral load (< 10⁶ copies/mL; n = 21) and high viral load (≥ 10⁶ copies/mL; n = 51). Cytokine concentrations are presented as mean ± SEM and were compared using Mann–Whitney U test. Exact p values are shown in the figure

Exploratory analyses identified significant associations between disease severity and age groups as well as liver enlargement (Table 1). Significant associations were also observed between viremia group and clinical symptoms, including vomiting and melena (Table S1). Furthermore, IL-10 levels were significantly associated with disease severity (Table 1), while IFN-γ, TNF-α, IL-8/CXCL8, and IL-10 levels were significantly associated with viremia groups (Table S1).

Table 1.

Association between clinical characteristics and disease severity among CHIKV-infected patients in Myanmar

	CHIKV +	Severe*	Non-severe*	P value
Age groups				0.009
≤ 5	24	18	6
6–15	43	27	16
16–45	5	0	5
Sex				0.382
Male	33	25	8
Female	39	20	19
Days of fever				0.494
1–2	11	8	3
3–4	38	25	13
≥ 5	17	6	11
Vomiting				0.069
Positive	42	31	11
Negative	25	14	11
Abdominal pain				0.055
Positive	13	11	2
Negative	51	31	20
Epistaxis				0.297
Positive	11	8	3
Negative	53	34	19
Malena				0.103
Positive	3	3	0
Negative	61	39	22
Liver enlargement				0.014
Positive	20	17	3
Negative	44	25	19
Platelet count (pg/ml)				0.294
Thrombocytopenia	19	14	5
Normal	45	30	15

*Disease severity was categorized according to the 2009 WHO dengue clinical classification

*Patients classified as dengue without warning signs (DWoWS) were grouped as non-severe CHIKV, whereas those classified as dengue with warning signs (DWWS) or severe dengue (SD) were grouped as severe CHIKV for exploratory analyses

IL-10 levels were significantly associated with disease severity (p = 0.040)

Because cytokine responses may vary over the course of infection, cytokine concentrations were also compared according to days of fever at presentation. No significant cytokine differences were observed among fever-duration groups, although TNF-α, IL-4, and IL-6 levels tended to decrease in patients with longer fever duration (Fig. S1).

Discussions

Our study identified cytokine responses in CHIKV-infected patients from Myanmar, including inflammatory and regulatory responses. Among the 10 cytokines included in the panel, 7 were detectable in serum, with significantly elevated IFN-γ, IL-4, IL-6, IL-8, IL-10, and GM-CSF levels observed in CHIKV-infected patients compared with healthy controls, supporting the activation of both inflammatory and regulatory immune responses during CHIKV infection. Similar cytokine elevations have been reported in CHIKV cohorts from Singapore, Sri Lanka, and other endemic settings [6–8]. The significantly elevated IL-6 and IL-8 levels observed in our study are consistent with inflammatory activation and leukocyte recruitment previously described in CHIKV infection, including a significant increase in GM-CSF, although this cytokine has been less consistently reported [6]. Together, these findings suggest that CHIKV infection in Myanmar induces measurable inflammatory, chemokine, growth factor, and regulatory cytokine responses similar to those reported in other geographic settings. IL-1β, IL-2, and IL-5 were not detected in the analyzed serum samples. As the present study was not designed to investigate the reasons for their non-detection, it was not possible to determine whether this reflected cytokine concentrations below the assay's measurable range, the timing of sample collection, or other biological or technical factors. CHIKV–DENV co-infected patients showed significantly higher IFN-γ and IL-10 levels than CHIKV mono-infected patients. Because CHIKV and DENV co-circulate in Myanmar and present with overlapping clinical manifestations, cytokine differences observed in co-infected patients remain relevant in endemic settings [3, 4 ]. Similar cytokine differences between CHIKV mono-infections and arboviral co-infections have been reported previously, suggesting that mixed arboviral infections may influence host immune responses [5, 9 ]. In our study, the significantly elevated IL-10 levels in the co-infected patients may reflect enhanced regulatory immune activity, whereas higher IFN-γ levels may indicate stronger antiviral immune activation. Nonetheless, these findings remain exploratory because of the limited number of co-infected cases. Viremia analysis demonstrated significantly higher TNF-α and IL-8 levels in patients with high viral load, whereas IFN-γ and IL-10 levels were significantly higher in patients with lower viral load, suggesting that cytokine responses did not increase uniformly with viral burden. Similar associations between cytokine responses and viral replication dynamics have been reported in CHIKV studies from India, Singapore, and Sri Lanka [6, 8, 10 ]. However, not all cytokine associations were identical across studies. For example, IL-8 was positively associated with high viral load in our cohort, whereas an opposite trend was reported in the Singapore and Sri Lanka cohorts, suggesting that cytokine response patterns may vary across endemic settings. In our study, higher TNF-α and IL-8 levels in high viral load patients, together with increased IFN-γ and IL-10 levels in low viral load patients, suggest that cytokine responses differ according to viral burden. However, because samples were collected at a single time point, these associations cannot be interpreted as causal or fully distinguished from temporal changes during infection. Exploratory analyses additionally identified significant associations between cytokine levels, disease severity, liver enlargement, viremia, and clinical manifestations. Increased IL-10 has previously been associated with regulatory immune responses and severe arboviral disease [7, 11 ]. Similar associations between cytokine responses, viral burden, and clinical manifestations have also been reported in CHIKV infection studies from Colombia, Brazil and India, even though the specific cytokines and clinical associations differed across studies [12, 13 ]. Together, these findings suggest that, although several cytokine responses were consistent with previous reports, differences in specific cytokine associations across studies highlight the importance of continued immunological characterization in different endemic settings. However, because of the exploratory nature of these analyses, causality cannot be established. Although, no significant cytokine differences were observed based on fever duration, TNF-α, IL-4, and IL-6 levels appeared to decrease with longer fever days, and this gradual decrease may reflect changes in cytokine levels over the course of infection. Distinct temporal changes in inflammatory cytokines during acute and later stages of CHIKV infection have also been described in previous studies [13]. This study had several limitations, including small subgroup sizes, a relatively small overall sample size, and a predominantly pediatric population. In addition, disease severity was stratified using the WHO 2009 dengue clinical classification as a pragmatic approach for exploratory analyses rather than a validated CHIKV severity classification. Therefore, associations between cytokine responses and disease severity should be interpreted cautiously. Furthermore, because this was a cross-sectional study, we could not follow how cytokine levels changed over time during infection or fully distinguish cytokine differences associated with viral load from those related to illness duration. Overall, these findings indicate that CHIKV infection in Myanmar is associated with measurable inflammatory and regulatory cytokine responses that vary according to viral load and clinical presentation.

Conclusions

This study identified distinct inflammatory and regulatory cytokine responses in CHIKV-infected patients from Myanmar, including variations according to viral load and clinical presentation. To the best of our knowledge, this is the first publicly available study describing cytokine profiles in CHIKV-infected patients from Myanmar. These findings expand the available immunological evidence from an understudied CHIKV-endemic setting and support further investigation of host immune responses during CHIKV infection.

Abbreviations

CHIKV

Chikungunya virus

DENV

Dengue virus

GM-CSF

Granulocyte–macrophage colony-stimulating factor

IFN-γ

Interferon-gamma

IL

Interleukin

RT–qPCR

Reverse transcription–quantitative polymerase chain reaction

TNF-α

Tumor necrosis factor-alpha

Author contributions

MK conceived the study, performed the laboratory investigations, analyzed the data, drafted and revised the manuscript. HL, KMN, KZT, and HMT contributed to sample collection and laboratory investigations. MN contributed to laboratory investigations. APW contributed to data analysis and manuscript revision. YN, YT, TU, and KM provided scientific guidance and critically reviewed the manuscript. MMNT conceived and supervised the study, secured funding, and critically revised the manuscript. All authors read and approved of the final manuscript.

Funding

This work was supported by the Japan Agency of Medical Research and Development (AMED) under grant number JP223fa627004, 24jm0210114h0001, 256f0137009j0001, wm0125011, wm0125006, and Japanese Society of Promotion and Science (JSPS) KAKENHI grant number 24K10246, 24K02288 and SDGs Research Project of Shimane University (30/2024). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

All data supporting the findings of this study are available within this paper and its Supplementary Information.

Declarations

Ethics approval and consent to participate

This study received clearance from the Ethics Review Committee on Medical Research Involving Human Subjects at the Department of Medical Research, Myanmar (Ethics/DMR/2017/068SE and 2020/110), as well as from the Institutional Review Board of the Institute of Tropical Medicine, Nagasaki University, Japan (191003233-3 and 230209288). Written informed consent was obtained from all participants and, where applicable, their legal guardians. Serum samples were collected under consent permitting future arboviral research and reuse; retrospective chikungunya analyses were approved under the existing protocol by both ethics’ committees.

Competing interests

The authors declare no competing interest.