Sequential determination of viral load, humoral responses and phylogenetic analysis in fatal and non-fatal cases of Crimean-Congo hemorrhagic fever patients from Gujarat, India, 2019

Abstract

Background: Thirty-four CCHF cases (17 fatal; 17 survived) were confirmed from Gujarat state, India during the year 2019. We aimed to find out the viral load, antibody kinetics, cytokine profile and phylogenetic analysis between fatal and non- fatal cases.

Methods: Thirty four cases were included in this study. Blood and urine samples were collected from all the cases on the day of admission to hospital. Non-fatal cases were followed weekly for understanding the profile of viral kinetics, anti-CCHFV IgM and IgG antibodies. We also quantified the cytokines in both fatal and non-fatal cases. For epidemiological correlation, livestock were screened for anti-CCHF IgG antibodies and the tick pool specimens were tested by real time RT-PCR. Virus isolation was attempted on tick pools and human specimens and phylogenetic analysis performed on human and ticks complete genome sequences.

Results: CCHF cases were detected throughout year in 2019 with the peak in August. Out of 34 cases, eight secondary CCHF cases were reported. Cases were predominantly detected in males and in 19-45 years age group (55.88%). The persistence of viremia was observed till 76th POD (post onset date) in one case whereas anti-CCHFV IgM and IgG was detected amongst these cases from the 2nd and 20th POD respectively. Positivity observed amongst livestock and tick pools were was 21.57% and 7.4% respectively. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. Fatal cases had high viral RNA copy numbers. Bleeding from one or two mucosal sites was significantly associated with fatality (OR-16.47;p-0.0034 at 95% CI). We could do CCHF virus isolation from two cases. Phylogenetic analysis revealed circulation of re-assortment of Asian-West African genotypes in humans and ticks.

Conclusions: The persistence of CCHF viral RNA was detected till 76th POD in one of the survivors. The circulation of a re-assortment Asian-West African genotype in a CCHF case is also reported first time from India.

Fig 1. District-wise CCHF positivity in the humans, tick-pools and livestock from Gujarat State, India during the year 2019.

Image attribution: https://d-maps.com/carte.php?num_car=8623&lang=en. License attribution: https://d-maps.com/conditions.php?lang=en. The map was modified showing the district-wise CCHF human cases, tick-pools and livestock positivity in the year 2019. All the details provided in the figure are created by the author themselves using the licensed version of the online software Biorender.com.

Fig 2. Month-wise distribution of CCHF cases and deaths in Gujarat state during 2019.

Fig 3. CCHF Viral RNA, anti-CCHFV IgM and anti-CCHFV IgG kinetics of CCHFV cases.

(A) CCHF viral RNA amongst fatal and survivors (B) CCHF viral RNA amongst individual survivors at different time points (C) Anti-CCHFV-IgM antibodies amongst individual survivors at different time points and fatal cases (D) Anti-CCHFV-IgG antibodies amongst individual survivors at different time points and fatal cases.

Fig 4. Level of Th1/Th2/Th17 cytokines in the serum samples of acute phase of infection in fatal cases (AFC) (n = 10), acute phase of infection in survived cases (ASC) (n = 13), recovered cases (RC) (n = 11) and healthy controls (HC) (n = 12) were determined using cytometric bead array (CBA) assay.

The results are expressed in scatter dot plot of individual values and data are given as Mean ± SEM (pg/ml) of (A) IL-6, (B) IL-10, (C) IFN-γ, (D) TNF-α and (E) IL-17A. Data were analyzed between groups by the non-parametric Kruskal–Wallis test followed by the post-hoc Dunn’s multiple comparison test. The horizontal lines indicate mean values. * P < 0.05; ** P < 0.01; *** P < 0.001.

Fig 5. Phylogenetic tree for the L M and S genes for the CCHFV (depicted in A, B and C respectively).

Maximum-Likelihood tree for CCHFV sequences retrieved from the clinical samples of the Gujarat state. The tree was constructed using the representative clades downloaded from Genbank using the General-time reversible model with gamma distribution as the rate parameter. The bootstrap replication of 1000 cycles was used to assess the statistical robustness of the generated tree. The scale depicts the number of base substitutions per site. Clades are marked in different colours. * indicates the fatal CCHF cases.