METHODS: To verify the causative agent of this outbreak and characterise the viral genes, the genes encoding the structural proteins C/prM/E of viruses isolated from local residents were sequenced followed by mutation and phylogenetic analysis. Recombination, selection pressure, potential secondary structure and three-dimensional structure analyses were also performed.
RESULTS: Phylogenetic analysis revealed that all epidemic strains were of the cosmopolitan DENV-2 genotype and were most closely related to the Zhejiang strain (MH010629, 2017) and then the Malaysia strain (KJ806803, 2013). Compared with the sequence of DENV-2SS, 151 base substitutions were found in the sequences of 89 isolates; these substitutions resulted in 20 non-synonymous mutations, of which 17 mutations existed in all samples (two in the capsid protein, six in the prM/M proteins, and nine in the envelope proteins). Moreover, amino acid substitutions at the 602nd (E322:Q → H) and 670th (E390: N → S) amino acids may have enhanced the virulence of the epidemic strains. One new DNA binding site and five new protein binding sites were observed. Two polynucleotide binding sites and seven protein binding sites were lost in the epidemic strains compared with DENV-2SS. Meanwhile, five changes were found in helical regions. Minor changes were observed in helical transmembrane and disordered regions. The 429th amino acid of the E protein switched from a histamine (positively charged) to an asparagine (neutral) in all 89 isolated strains. No recombination events or positive selection pressure sites were observed. To our knowledge, this study is the first to analyse the genetic characteristics of epidemic strains in the first dengue outbreak in Hunan Province in inland China.
CONCLUSIONS: The causative agent is likely to come from Zhejiang Province, a neighbouring province where dengue fever broke out in 2017. This study may help clarify the intrinsic geographical relatedness of DENV-2 and contribute to further research on pathogenicity and vaccine development.
METHODS: Over six months in 2018, we recruited 368 adults who met the WHO 2009 criteria for probable dengue infection. They underwent the following blood tests: full blood count, dengue virus (DENV) rapid diagnostic test (RDT), ELISA (dengue IgM and IgG), nested RT-PCR for dengue, multiplex qRT-PCR for Zika, Chikungunya and dengue as well as PCR tests for Leptopspira spp., Japanese encephalitis and West Nile virus.
RESULTS: Laboratory-confirmed dengue infections (defined by positive tests in NS1, IgM, high-titre IgG or nested RT-PCR) were found in 167 (45.4%) patients. Of these 167 dengue patients, only 104 (62.3%) were positive on rapid diagnostic testing. Dengue infection was significantly associated with the following features: family or neighbours with dengue in the past week (AOR: 3.59, 95% CI:2.14-6.00, p<0.001), cutaneous rash (AOR: 3.58, 95% CI:1.77-7.23, p<0.001), increased temperature (AOR: 1.33, 95% CI:1.04-1.70, p = 0.021), leucopenia (white cell count < 4,000/μL) (AOR: 3.44, 95% CI:1.72-6.89, p<0.001) and thrombocytopenia (platelet count <150,000/μL)(AOR: 4.63, 95% CI:2.33-9.21, p<0.001). Dengue infection was negatively associated with runny nose (AOR: 0.47, 95% CI:0.29-0.78, p = 0.003) and arthralgia (AOR: 0.42, 95% CI:0.24-0.75, p = 0.004). Serotyping by nested RT-PCR revealed mostly mono-infections with DENV-2 (n = 64), DENV-1 (n = 32) and DENV-3 (n = 17); 14 co-infections occurred with DENV-1/DENV-2 (n = 13) and DENV-1/DENV-4 (n = 1). Besides dengue, none of the pathogens above were found in patients' serum.
CONCLUSIONS: Acute undifferentiated febrile infections are a diagnostic challenge for community-based clinicians. Rapid diagnostic tests are increasingly used to diagnose dengue infection but negative tests should be interpreted with caution as they fail to detect a considerable proportion of dengue infection. Certain clinical features and haematological parameters are important in the clinical diagnosis of dengue infection.
Methods: Each 24-well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR-12. Following 48 h of incubation, inhibitory efficacies of AR-12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT-PCR. The underlying mechanism(s) possibly involved in the compound's inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains.
Results: The qRT-PCR data demonstrated that DENV-3 was most potently inhibited by AR-12, followed by DENV-1, DENV-2 and DENV-4. Our molecular docking findings suggested that AR-12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins.
Conclusions: These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR-12 as a potential therapeutic option for DENV infection.