Molecular and Entomological Characterization of 2023 Dengue Outbreak in Dhading District, Central Nepal.

In 2023, Nepal faced its second largest dengue outbreak ever, following a record-breaking number of dengue cases in 2022, characterized by the expansion of infections into areas of higher altitudes. However, the characteristics of the 2023 circulating dengue virus (DENV) and the vector density remain poorly understood. Therefore, we performed DENV serotyping, clinical and laboratory assessment, and entomological analysis of the 2023 outbreak in central Nepal. A total of 396 fever cases in Dhading hospital suspected of being DENV positive were enrolled, and blood samples were collected and tested by different techniques including PCR. Of these, 278 (70.2%) had confirmed DENV infection. Multiple serotypes (DENV-1, -2, and -3) were detected. DENV-2 (97.5%) re-emerged after six years in Dhading while DENV-3 was identified for the first time. Dengue inpatients had significantly higher frequency of anorexia, myalgia, rash, diarrhea, nausea, vomiting, abdominal pain, and thrombocytopenia (p < 0.05). In this area, Aedes mosquitoes largely predominated (90.7%) with the majority being A. aegypti (60.7%). We also found high levels of Aedes index (20.0%) and container index (16.7%). We confirmed multiple DENV serotype circulation with serotype re-emergence and new serotype introduction, and high vector density in 2023. These findings call for the urgent initiation and scaling up of DENV molecular surveillance in human and mosquito populations for dengue control and prevention in Nepal.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Possible vertical transmission of Chikungunya virus infection detected in the cord blood samples from a birth cohort in Vietnam.

BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus (genus Alphavirus, family Togaviridae) that is primarily transmitted to humans by Aedes mosquitoes, and can be transmitted from mother to child. Little is known about CHIKV transmission in Vietnam, where dengue is endemic and Aedes mosquitoes are abundant. This study aimed to determine the prevalence and characteristics of vertical CHIKV infection in a birth cohort, and seroprevalence of anti-CHIKV antibodies with or without confirmation by neutralization tests among women bearing children in Vietnam. METHODS: We collected umbilical cord blood plasma samples from each newly delivered baby in Nha Trang, Central Vietnam, between July 2017 and September 2018. Samples were subjected to molecular assay (quantitative real-time RT-PCR) and serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assay, and neutralization tests). RESULTS: Of the 2012 tested cord blood samples from newly delivered babies, the CHIKV viral genome was detected in 6 (0.3%) samples by RT-PCR, whereas, 15 samples (0.7%) were anti-CHIKV-IgM positive. Overall, 18 (0.9%, 95% CI: 0.6-1.5) samples, including three positives for both CHIKV IgM and viral genome on RT-PCR, were regarded as vertical transmission of CHIKV infection. Of the 2012 cord blood samples, 10 (0.5%, 95% CI: 0.2-0.9) were positive for both anti-CHIKV IgM and IgG. Twenty-nine (1.4%, 95% CI: 1.0-2.1) were seropositive for anti-CHIKV IgG while 26 (1.3%, 95% CI: 0.8-1.9) of them were also positive for neutralizing antibodies, and regarded as seropositive with neutralization against CHIKV infection. CONCLUSION: This is the first report of a possible CHIKV maternal-neonatal infection in a birth cohort in Vietnam. The findings indicate that follow-up and a differential diagnosis of CHIKV infection in pregnant women are needed to clarify the potential for CHIKV vertical transmission and its impact in the newborn.

Role of pre-existing immunity in driving the dengue virus serotype 2 genotype shift in the Philippines: A retrospective analysis of serological data.

OBJECTIVE: The invasion of dengue virus (DENV)-2 Cosmopolitan genotype into the Philippines, where the Asian II genotype previously circulated challenges the principle of dengue serotype-specific immunity. Assessment of antibodies in this population may provide a mechanistic basis for how new genotypes emerge in dengue-endemic areas. METHODS: We evaluated the neutralizing antibody (nAb) and antibody-dependent enhancement (ADE) responses against the two genotypes using archived serum samples collected from 333 patients with confirmed dengue in Metro Manila, Philippines, before, during, and after the introduction of the Cosmopolitan genotype. We quantified nAb titers in baby hamster kidney (BHK-21) cells with or without the Fcγ receptor IIA (FcγRIIA) to detect the capacity of virus-antibody complexes to neutralize or enhance DENV. RESULTS: The nAb potency of the archived serum samples against the two genotypes was greatly affected by the presence of FcγRIIA. We found significant differences in nAb titers between the two genotypes in BHK-21 cells with FcγRIIA (P <0.0001). The archived serum samples were incapable of fully neutralizing the Cosmopolitan genotype, but instead strongly promoted its ADE compared to the Asian II genotype (P <0.0001). CONCLUSION: These results reinforce the role of pre-existing immunity in driving genotype shifts. Our finding that specific genotypes exhibit differing susceptibilities to ADE by cross-reactive antibodies may have implications for dengue vaccine development.

Suppression of dengue virus replication by the French maritime pine extract Pycnogenol®.

Dengue virus (DENV) is mainly found in the tropics and infects approximately 400 million people annually. However, no clinically available therapeutic agents specific to dengue have been developed. Here, we examined the potential antiviral effects of the French maritime pine extract Pycnogenol® (PYC) against DENV because we previously found that the extract exerts antiviral effects on hepatitis C virus, which belongs to the Flavivirus family. First, we examined the efficacy of PYC against DENV1, 2, 3, and 4 serotypes and determined that it had a dose-dependent suppressive effect on the viral load, especially in the supernatant. This inhibitory effect of PYC may target the late stages of infection such as maturation and secretion, but not replication. Next, we examined the efficacy of PYC against DENV infection in type I interferon (IFN) receptor knockout mice (A129). As the propagation of DENV2 was the highest among the four serotypes, we used this serotype in our murine model experiments. We found that PYC significantly inhibited DENV2 replication in mice on day 4 without significantly decreasing body weight or survival ratio. We further examined the mechanism of action of PYC in DENV2 infection by characterizing the main PYC targets among the host (viral) factors and silencing them using siRNA. Silencing long noncoding-interferon-induced protein (lnc-IFI)-44, polycystic kidney disease 1-like 3 (Pkd1l3), and ubiquitin-specific peptidase 31 (Usp31) inhibited the replication of DENV2. Thus, the results of this study shed light on the inhibitory effects of PYC on DENV replication and its underlying mechanisms.

A Novel, Comprehensive A129 Mouse Model for Investigating Dengue Vaccines and Evaluating Pathogenesis.

In search of a mouse model for use in evaluating dengue vaccines, we assessed A129 mice that lacked IFN-α/ß receptors, rendering them susceptible to dengue virus (DENV) infection. To our knowledge, no reports have evaluated dengue vaccine efficiency using A129 mice. A129 mice were given a single intraperitoneal (IP) or subcutaneous (SC) injection of the vaccine, Dengvaxia. After 14 days of immunization via the IP or SC injection of Dengvaxia, the A129 mice exhibited notably elevated levels of anti-DENV immunoglobulin G and neutralizing antibodies (NAb) targeting all four DENV serotypes, with DENV-4 displaying the highest NAb levels. After challenge with DENV-2, Dengvaxia and mock-immunized mice survived, while only the mock group exhibited signs of morbidity. Viral genome levels in the serum and tissues (excluding the brain) were considerably lower in the immunized mice compared to those in the mock group. The SC administration of Dengvaxia resulted in lower viremia levels than IP administration did. Therefore, given that A129 mice manifest dengue-related morbidity, including viremia in the serum and other tissues, these mice represent a valuable model for investigating novel dengue vaccines and antiviral drugs and for exploring dengue pathogenesis.

An outbreak of a novel lineage of dengue virus 2 in Vietnam in 2022.

In 2022, a large dengue outbreak was reported in Vietnam, where dengue was endemic. A total of 1889 acute-phase serum samples were collected from patients with suspected dengue at Vung Tau General Hospital, the core hospital in Vung Tau Province, southern Vietnam. Among the 1889 samples analyzed for laboratory confirmation of dengue virus (DENV) infection, 339 positive cases were identified, of which 130 were primary infections and 209 were secondary infections. DENV-2 was the dominant serotype in both primary and secondary infection groups. Phylogenetic analysis based on sequences of the envelope protein-coding region revealed the emergence of a new DENV-2 lineage during this outbreak.

Serological and Molecular Epidemiology of Chikungunya Virus Infection in Vietnam, 2017-2019.

Chikungunya fever is an acute febrile illness caused by the chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Since 1965, only a few studies with limited scope have been conducted on CHIKV in Vietnam. Thus, this study aimed to determine the seroprevalence and molecular epidemiology of CHIKV infection among febrile patients in Vietnam from 2017 to 2019. A total of 1063 serum samples from 31 provinces were collected and tested for anti-CHIKV IgM and IgG ELISA. The 50% focus reduction neutralization test (FRNT50) was used to confirm CHIKV-neutralizing antibodies. Quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the presence of the CHIKV genome. The results showed that 15.9% (169/1063) of the patients had anti-CHIKV IgM antibodies, 20.1% (214/1063) had anti-CHIKV IgG antibodies, 10.4% (111/1063) had CHIKV-neutralizing antibodies, and 27.7% (130/469) of the samples were positive in RT-qPCR analysis. The E1 CHIKV genome sequences were detected among the positive RT-qPCR samples. Our identified sequences belonged to the East/Central/South/African (ECSA) genotype, which has been prevalent in Vietnam previously, suggesting CHIKV has been maintained and is endemic in Vietnam. This study demonstrates a high prevalence of CHIKV infection in Vietnam and calls for an annual surveillance program to understand its impact.

A Birth Cohort Follow-Up Study on Congenital Zika Virus Infection in Vietnam.

We assessed the development, sensory status, and brain structure of children with congenital Zika virus (ZIKV) infection (CZI) at two years and preschool age. CZI was defined as either ZIKV RNA detection or positive ZIKV IgM and neutralization test in the cord or neonatal blood. Twelve children with CZI born in 2017-2018 in Vietnam, including one with Down syndrome, were assessed at 23-25.5 months of age, using Ages and Stages Questionnaire (ASQ-3), ASQ:Social-Emotional (ASQ:SE-2), Modified Checklist for Autism in Toddlers, automated auditory brainstem response (AABR), and Spot Vision Screener (SVS). They underwent brain CT and MRI. They had detailed ophthalmological examinations, ASQ-3, and ASQ:SE-2 at 51-62 months of age. None had birthweight or head circumference z-score < -3 except for the one with Down syndrome. All tests passed AABR (n = 10). No ophthalmological problems were detected by SVS (n = 10) and detailed examinations (n = 6), except for a girl's astigmatism. Communication and problem-solving domains in a boy at 24 months, gross-motor area in a boy, and gross-motor and fine-motor areas in another boy at 59-61 months were in the referral zone. Brain CT (n = 8) and MRI (n = 6) revealed no abnormalities in the cerebrum, cerebellum, or brainstem other than cerebellar hypoplasia with Down syndrome. The CZI children were almost age-appropriately developed with no brain or eye abnormalities. Careful and longer follow-up is necessary for children with CZI.

Clinical, Virological, and Immunological Features in Cosmopolitan Genotype DENV-2-Infected Patients during a Large Dengue Outbreak in Sri Lanka in 2017.

In 2017, Sri Lanka experienced its largest dengue epidemic and reported severe and unusual presentations of dengue with high morbidity. This outbreak was associated with the reemergence of dengue virus-2 (DENV-2), with the responsible strain identified as a variant of the previously circulating DENV-2 cosmopolitan genotype. In this study, we characterized the DENV-2 cosmopolitan genotype from patients during this epidemic. Also, we identified host factors that contributed to the severity of dengue infection in patients infected with this particular virus. Ninety-one acute serum samples from patients at the National Hospital in Kandy were randomly selected. Of these, 40.2% and 48.9% were positive for dengue IgM and IgG, respectively. NS1 antigen levels were significantly higher in primary infections. The severe dengue (SD) and dengue with warning signs (DWWS) groups exhibited significantly higher viral genome and infectivity titers than the dengue without warning signs (DWoWS) group. The highest viremia level was observed in SD patients. As for host cytokine response, interferon α (IFN-α) levels were significantly higher in the DWoWS group than in the DWWS and SD groups, whereas interleukin (IL)-12p40 and tumor necrosis factor α (TNF-α) levels in SD patients were significantly higher than in the other two groups. The TNF-α, IL-4, and monocyte chemoattractant protein-1 concentrations were positively correlated with NS1 antigen levels. From whole-genome analysis, NS4 had the highest frequency of amino acid variants, followed by the E gene. Our study suggests that viremia levels and immune responses contributed to SD outcomes, and these findings may help in identifying an effective therapeutic strategy against SD infection.

Burden of Chikungunya Virus Infection during an Outbreak in Myanmar.

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central South African genotype of CHIKV occurred in Myanmar, and during this period, a cross-sectional study was conducted in two regions, Mandalay and Yangon, to examine the molecular and seropositivity rate of the CHIKV infection. The participants (1124) included dengue-suspected pediatric patients, blood donors, and healthy volunteers, who were assessed using molecular assays (quantitative real-time RT-PCR), serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assays), and neutralization tests. The tests confirmed the following positivity rates: 11.3% (127/1124) for the molecular assay, 12.4% (139/1124) for the anti-CHIKV IgM Ab, 44.5% (500/1124) for the anti-CHIKV IgG Ab, and 46.3% (520/1124) for the CHIKV neutralizing Ab. The highest rate for the molecular test occurred with the dengue-suspected pediatric patients. The seroprevalence rate through natural infection was higher in the healthy volunteers and blood donors than that in the pediatric patients. The results of this study will help stakeholders determine the criteria for choosing appropriate recipients when a CHIKV vaccine is introduced in Myanmar.

Epidemiological evidence of acute transmission of Zika virus infection in dengue suspected patients in Sri-Lanka.

BACKGROUND: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. METHODS: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. RESULTS: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. CONCLUSION: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these viruses.

Coinfection and circulation of chikungunya virus and dengue virus in pediatric patients in Myanmar, 2019.

Myanmar is an endemic country for arboviruses, and outbreaks occur frequently. A cross-sectional analytical study was conducted during the peak season of the chikungunya virus (CHIKV) outbreak in 2019. A total of 201 patients with acute febrile illness who were admitted to the 550-bedded Mandalay Children Hospital in Myanmar were enrolled in the study, and virus isolation, serological tests, and molecular tests for the dengue virus (DENV) and CHIKV were performed for all samples. Out of 201 patients, 71 (35.3%) were only DENV-infected, 30 (14.9%) were only CHIKV-infected and 59 (29.4%) were coinfected with DENV and CHIKV. The viremia levels of the DENV- and CHIKV- mono-infected groups were significantly higher than those of the group coinfected with DENV and CHIKV. Genotype I of DENV-1, genotypes I and III of DENV-3, genotype I of DENV-4 and the East/Central/South African genotype of CHIKV were co-circulating during the study period. Two novel epistatic mutations of CHIKV (E1:K211E and E2:V264A) were noted. This study highlighted that there were many coinfection cases during the outbreak and that the co-circulation of both viruses in DENV-endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance to facilitate the implementation of effective control measures.

Co-Circulation of Dengue Virus Serotypes 1, 2, and 3 during the 2022 Dengue Outbreak in Nepal: A Cross-Sectional Study.

The largest dengue outbreak in the history of Nepal occurred in 2022, with a significant number of casualties. It affected all 77 districts, with the nation's capital, Kathmandu (altitude 1300 m), being the hardest hit. However, the molecular epidemiology of this outbreak, including the dengue virus (DENV) serotype(s) responsible for this epidemic, remain unknown. Here, we report the epidemic trends, clinico-laboratory features, and virus serotypes and their viral load profiles that are associated with this outbreak in Nepal. Dengue-suspected febrile patients were investigated by routine laboratory, serological, and molecular tools, including a real-time quantitative polymerase chain reaction (qRT-PCR). Of the 538 dengue-suspected patients enrolled, 401 (74.5%) were diagnosed with dengue. Among these dengue cases, 129 (32.2%) patients who required hospital admission had significant associations with myalgia, rash, diarrhea, retro-orbital pain, bleeding, and abdominal pain. DENV-1, -2, and -3 were identified during the 2022 epidemic, with a predominance of DENV-1 (57.1%) and DENV-3 (32.1%), exhibiting a new serotype addition. We found that multiple serotypes circulated in 2022, with a higher frequency of hospitalizations, more severe dengue, and more deaths than in the past. Therefore, precise mapping of dengue and other related infections through integrated disease surveillance, evaluation of the dynamics of population-level immunity and virus evolution should be the urgent plans of action for evidence-based policy-making for dengue control and prevention in the country.

Core fucose-specific Pholiota squarrosa lectin (PhoSL) as a potent broad-spectrum inhibitor of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.

Has COVID-19 suppressed dengue transmission in Nepal?

Following the report of the first COVID-19 case in Nepal on 23 January 2020, three major waves were documented between 2020 and 2021. By the end of July 2022, 986 596 cases of confirmed COVID-19 and 11 967 deaths had been reported and 70.5% of the population had received at least two doses of a COVID-19 vaccine. Prior to the pandemic, a large dengue virus (DENV) epidemic affected 68 out of 77 districts, with 17 932 cases and six deaths recorded in 2019. In contrast, the country's Epidemiology and Disease Control Division reported 530 and 540 dengue cases in the pandemic period (2020 and 2021), respectively. Furthermore, Kathmandu reported just 63 dengue cases during 2020 and 2021, significantly lower than the 1463 cases reported in 2019. Serological assay showed 3.2% positivity rates for anti-dengue immunoglobulin M antibodies during the pandemic period, contrasting with 26.9-40% prior to it. Real-time polymerase chain reaction for DENV showed a 0.5% positive rate during the COVID-19 pandemic which is far lower than the 57.0% recorded in 2019. Continuing analyses of dengue incidence and further strengthening of surveillance and collaboration at the regional and international levels are required to fully understand whether the reduction in dengue incidence/transmission were caused by movement restrictions during the COVID-19 pandemic.

How an Outbreak of COVID-19 Circulated Widely in Nepal: A Chronological Analysis of the National Response to an Unprecedented Pandemic.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The first COVID-19 case was reported in Wuhan, China, in December 2019. In March 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic. The first COVID-19 case in Nepal was reported in January 2020 in a Nepalese man who had returned from Wuhan to Nepal. This study aims to evaluate the government of Nepal's (GoN) response to the COVID-19 pandemic and explore ways to prevent COVID-19 and other pandemic diseases in the future. As of May 2022, a total of 979,140 cases and 11,951 deaths associated with COVID-19 have been reported in Nepal. To prevent the spread of the virus, the GoN initiated various preventive and control measures, including lockdown strategies. The effects of COVID-19 are expected to persist for many years; the best strategies a resource-limited country such as Nepal can implement to control pandemic diseases such as COVID-19 in the pre-vaccine stage are to increase testing, tracing, and isolation capacity.