Anti-Arbovirus Antibodies Cross-React With Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is found in regions where dengue (DENV) and chikungunya (CHIKV) viruses are endemic. Any serological cross-reactivity between DENV, CHIKV, and SARS-CoV-2 is significant as it could lead to misdiagnosis, increased severity, or cross-protection. This study examined the potential cross-reactivity of anti-DENV and CHIKV antibodies with SARS-CoV-2 using acute and convalescent-phase samples collected before the SARS-CoV-2 pandemic. These included healthy, normal human (NHS, n = 6), CHIKV-positive (n = 14 pairs acute and convalescent), primary DENV-positive (n = 20 pairs), secondary DENV-positive (n = 20 pairs), and other febrile illnesses sera (n = 23 pairs). Samples were tested using an in-house SARS-CoV-2 and a EUROIMMUN IgA and IgG ELISAs. All NHS samples were negative, whereas 3.6% CHIKV, 21.7% primary DENV, 15.7% secondary DENV, and 10.8% febrile diseases sera resulted as anti-SARS-CoV-2 antibody positive. The EUROIMMUN ELISA using spike 1 as the antigen detected more positives among the primary DENV infections than the in-house ELISA using spike 1-receptor binding domain (RBD) protein. Among ELISA-positive samples, four had detectable neutralizing antibodies against SARS-CoV-2 reporter virus particles yet none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated the SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. IMPORTANCE SARS-CoV-2 continues to cause significant morbidity globally, including in areas where DENV and CHIKV are endemic. Reports using rapid diagnostic and ELISAs have demonstrated that serological cross-reactivity between DENV and SARS-CoV-2 can occur. Furthermore, it has been observed that convalescent DENV patients are at a lower risk of developing COVID-19. This phenomenon can interfere with the accuracy of serological testing and clinical management of both DENV and COVID-19 patients. In this study, the cross-reactivity of primary/secondary anti-DENV, CHIKV, and other febrile illness antibodies with SARS-CoV-2 using two ELISAs has been shown. Among ELISA-positive samples, four had detectable levels of neutralizing antibodies against SARS-CoV-2 reporter virus particles. However, none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. The data discussed here provide information regarding diagnosis and may help guide appropriate public health interventions.

Comparative Analyses of Historical Trends in Confirmed Dengue Illnesses Detected at Public Hospitals in Bangkok and Northern Thailand, 2002-2018.

Dengue is a re-emerging global public health problem, the most common arbovirus causing human disease in the world, and a major cause of hospitalization in endemic countries causing significant economic burden. Data were analyzed from passive surveillance of hospital-attended dengue cases from 2002 to 2018 at Phramongkutklao Hospital (PMKH) located in Bangkok, Thailand, and Kamphaeng Phet Provincial Hospital (KPPH) located in the lower northern region of Thailand. At PMKH, serotype 1 proved to be the most common strain of the virus, whereas at KPPH, serotypes 1, 2, and 3 were the most common strains from 2006 to 2008, 2009 to 2012, and 2013 to 2015, respectively. The 11-17 years age-group made up the largest proportion of patients impacted by dengue illnesses during the study period at both sites. At KPPH, dengue virus (DENV)-3 was responsible for most cases of dengue fever (DF), whereas it was DENV-1 at PMKH. In cases where dengue hemorrhagic fever was the clinical diagnosis, DENV-2 was the predominant serotype at KPPH, whereas at PMKH, it was DENV-1. The overall disease prevalence remained consistent across the two study sites with DF being the predominant clinical diagnosis as the result of an acute secondary dengue infection, representing 40.7% of overall cases at KPPH and 56.8% at PMKH. The differences seen between these sites could be a result of climate change increasing the length of dengue season and shifts in migration patterns of these populations from rural to urban areas and vice versa.

Molecular epidemiology of dengue fever outbreaks in Bhutan, 2016-2017.

Dengue continues to pose a significant public health problem in tropical and subtropical countries. In Bhutan, first outbreak of dengue fever (DF) was reported in 2004 in a southern border town, followed by sporadic cases over the years. In this study, we analysed DF outbreaks that occurred in 3 different places during the years 2016 and 2017. A total of 533 cases in 2016 and 163 in 2017 were suspected of having of DF, where young adults were mostly affected. A total of 240 acute serum specimens collected and analyzed for serotype by nested RT-PCR revealed predominance of serotypes 1 and 2 (DENV-1 and 2). Phylogenetic analysis using envelope gene for both the serotypes demonstrated cosmopolitan genotype which were closely related to strains from India, indicating that they were probably imported from the neighboring country over the past few years.

Chikungunya Virus Infections Among Patients with Dengue-Like Illness at a Tertiary Care Hospital in the Philippines, 2012-2013.

Chikungunya virus (CHIKV) often co-circulates with dengue virus (DENV). A cross-sectional surveillance study was conducted at a tertiary hospital in Manila, Philippines, to describe the prevalence and characteristics of DENV and CHIKV infections among patients seeking care for dengue-like illness. Acute blood samples from patients ≥ 6 months of age clinically diagnosed with dengue from November 2012 to December 2013 underwent reverse transcription polymerase chain reaction (RT-PCR) to detect DENV and CHIKV RNA. A total of 118 patients with clinically diagnosed dengue (age range = 1-89 years, mean = 22 years; male-to-female ratio = 1.51) were tested by DENV RT-PCR; 40 (34%) were DENV PCR-positive (age range = 1-45 years, mean = 17 years). All DENV serotypes were detected: 11 (28%) DENV-1, 6 (15%) DENV-2, 6 (15%) DENV-3, and 17 (42%) DENV-4. Of 112 patients clinically diagnosed with dengue and tested by CHIKV RT-PCR, 11 (10%) were CHIKV PCR-positive (age range = 2-47 years, mean = 20.3 years). No coinfections were detected. Presenting signs/symptoms did not differ between DENV- and CHIKV-positive cases. Sequencing of envelope 1 gene from two CHIKV PCR-positive samples showed Asian genotype. This study highlights the potential for misdiagnosis of medically attended CHIKV infections as DENV infection and the difficulty in clinically differentiating dengue and chikungunya based on presenting signs/symptoms alone. This underscores the necessity for diagnostic laboratory tests to distinguish CHIKV infections in the background of actively co-circulating DENV.

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

BACKGROUND: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. OBJECTIVE: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. STUDY DESIGN: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. RESULTS AND CONCLUSION: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.

Comparative analysis of full-length genomic sequences of 10 dengue serotype 1 viruses associated with different genotypes, epidemics, and disease severity isolated in Thailand over 22 years.

Comparative sequence analysis was performed on the full-length genomic sequences of 10 representative dengue virus serotype 1 (DENV-1) strains sampled from patients at Children's Hospital, Bangkok, Thailand over a 22-year period, which represented different epidemics, disease severity, and sampling time. The results showed remarkable inter-genotypic variation between predominant and non-predominant genotypes and genotype-specific amino acids and nucleotides throughout the entire viral genome except for the 5'-non-translated region. The frequency of intra-genotypic variation was correlated with dengue transmission rate and sampling time. The 5'-non-translated region of all 10 viruses was highly conserved for predominant and non-predominant genotypes and NS2B was the most conserved protein. Some intra-genotypic substitutions of amino acids and nucleotides in predominant genotype strains were fixed in the viral genome since 1994, which indicated that the evolution of predominant genotype strains in situ over time might contribute to increased virus fitness important for sustaining dengue epidemics in Thailand.

Microevolution of Dengue viruses circulating among primary school children in Kamphaeng Phet, Thailand.

To determine the extent and structure of genetic variation in dengue viruses (DENV) on a restricted spatial and temporal scale, we sequenced the E (envelope) genes of DENV-1, -2, and -3 isolates collected in 2001 from children enrolled in a prospective school-based study in Kamphaeng Phet, Thailand, and diagnosed with dengue disease. Our analysis revealed substantial viral genetic variation in both time and space, with multiple viral lineages circulating within individual schools, suggesting the frequent gene flow of DENV into this microenvironment. More-detailed analyses of DENV-2 samples revealed strong clustering of viral isolates within individual schools and evidence of more-frequent viral gene flow among schools closely related in space. Conversely, we observed little evolutionary change in those viral isolates sampled over multiple time points within individual schools, indicating a low rate of mutation fixation. These results suggest that frequent viral migration into Kamphaeng Phet, coupled with population (school) subdivision, shapes the genetic diversity of DENV on a local scale, more so than in situ evolution within school catchment areas.

Population genetic structure of Anopheles maculatus in Thailand.

Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.

Structure and age of genetic diversity of dengue virus type 2 in Thailand.

Dengue virus type 2 (DENV-2) is a common viral infection and an important health concern in South-East Asia. To determine the molecular evolution of DENV-2 in Thailand, 105 isolates of the E (envelope) gene and 10 complete genomes sampled over a 27 year period were sequenced. Phylogenetic analysis of these data revealed that three genotypes of DENV-2 have circulated in Thailand, although, since 1991, only viruses assigned to Asian genotype I have been sampled from the population. A broader analysis of 35 complete genomes of DENV-2 revealed that most amino acids are subject to strong selective constraints, indicative of widespread purifying selection against deleterious mutations. This was further supported by an analysis of genome-wide substitution rates, which indicated that DENV-2 fixes approximately 10 mutations per genome per year, far lower than expected from its mutational dynamics. Finally, estimates of the age of DENV-2 were remarkably consistent among genes, indicating that the current genetic diversity in this virus probably arose within the last 120 years, concordant with the first determination of the aetiology of dengue disease.

Clade replacements in dengue virus serotypes 1 and 3 are associated with changing serotype prevalence.

The evolution of dengue virus (DENV) is characterized by phylogenetic trees that have a strong temporal structure punctuated by dramatic changes in clade frequency. To determine the cause of these large-scale phylogenetic patterns, we examined the evolutionary history of DENV serotype 1 (DENV-1) and DENV-3 in Thailand, where gene sequence and epidemiological data are relatively abundant over a 30-year period. We found evidence for the turnover of viral clades in both serotypes, most notably in DENV-1, where a major clade replacement event took place in genotype I during the mid-1990s. Further, when this clade replacement event was placed in the context of changes in serotype prevalence in Thailand, a striking pattern emerged; an increase in DENV-1 clade diversity was associated with an increase in the abundance of this serotype and a concomitant decrease in DENV-4 prevalence, while clade replacement was associated with a decline in DENV-1 prevalence and a rise of DENV-4. We postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses.

Cytochrome P450 genes: molecular cloning and overexpression in a pyrethroid-resistant strain of Anopheles minimus mosquito.

We previously determined that physiological resistance in a laboratory-selected pyrethroid-resistant Anopheles minimus species A Theobald mosquito is associated with increased detoxification via a P450-mediated mechanism. A CYP6 gene, CYP6AA3, was subsequently cloned and found overexpressed in 2 resistant mosquito generations (F13 and F19). We report herein the cloning of CYP6P7 and CYP6P8 genes with full coding sequences from the same An. minimus mosquito colony strain. CYP6P7 and CYP6P8 encode proteins, each with 509 amino acids. CYP6P7 had the closest (81%) amino acid identity with Anopheles gambiae CYP6P2. CYP6P8 genes had 79% identity with An. gambiae CYP6P1. Using semiquantitative reverse transcription-polymerase chain reaction analysis, the mRNA expression level of CYP6P7 presented approximately 2- and 4-fold increases in F19 and F25 deltamethrin-resistant populations, respectively, compared with the parent susceptible strain. CYP6P8 mRNA expression levels were not significantly different between the 3 filial generations. The overexpression of CYP6AA3 mRNA was greater than that of CYP6P7 in F19 and F25 resistant populations. The relative increase of both CYP6AA3 and CYP6P7 mRNA was correlated with increased resistance to deltamethrin in An. minimus.

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.