Novel Antiviral Efficacy of Hedyotis diffusa and Artemisia capillaris Extracts against Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Infection and Immunoregulatory Cytokine Signatures

Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose–responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications

Stability and Infectivity of SARS-CoV-2 and Viral RNA in Water, Commercial Beverages, and Bodily Fluids

The stability and infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in liquid samples are of great concern to virus transmission via common beverages and sewage water. Here, we investigated the stability of SARS-CoV-2 in 32 liquids including common beverages, bodily fluids, and commonly used viral transport media. Our results showed that the infectious virus could be recovered up to 77-days from common beverages including milk and water. Viral RNA could be detected at high levels in all samples up to 28-days, indicating that while viral RNA demonstrates higher stability than infectivity, viral RNA levels do not reflect the infectious capability of SARS-CoV-2. These results indicate that SARS-CoV-2 is highly stable in optimal conditions and a sufficient control measure is needed in reducing the risk of exposure and controlling and preventing future outbreaks

Importation of Dengue Virus Type 3 to Japan from Tanzania and Côte d’Ivoire

Travelers can introduce viruses from disease-endemic to non–disease-endemic areas. Serologic and virologic tests confirmed dengue virus infections in 3 travelers returning to Japan: 2 from Tanzania and 1 from Côte d’Ivoire. Phylogenetic analysis of the envelope gene showed that 2 genetically related virus isolates belonged to dengue virus type 3 genotype III

Characteristics of COVID-19 epidemic and control measures to curb transmission in Malaysia

The first wave of COVID-19 epidemic began in late January in Malaysia and ended with a very small size. The second wave of infections broke out in late February and grew rapidly in the first 3 weeks. Authorities in the country responded quickly with a series of control strategies collectively known as the Movement Control Order (MCO) with different levels of intensity matching the progression of the epidemic. We examined the characteristics of the second wave and discussed the key control strategies implemented in the country. In the second wave, the epidemic doubled in size every 3.8 days (95% confidence interval: 3.3, 4.5) in the first month and decayed slowly after that with a halving time of approximately 3 weeks. The time-varying reproduction number Rt peaked at 3.1 (95% credible interval: 2.7, 3.5) in the 3rd week, declined sharply thereafter and stayed below 1 in the last 3 weeks of April,indicating low transmissibility approximately 3 weeks after the MCO. The experience of Malaysia suggests that adaptive triggering of distancing policies combined with a population-wide movement control measure can be effective in suppressing transmission and preventing a rebound

Dengue Virus Infection-Enhancing Activity in Serum Samples with Neutralizing Activity as Determined by Using FcγR-Expressing Cells

Dengue has become a major international public health concern in recent decades. There are four dengue virus serotypes. Recovery from infection with one serotype confers life-long protection to the homologous serotype but only partial protection to subsequent infection with other serotypes. Secondary infection with a serotype different from that in primary infection increases the risk of development of severe complications. Antibodies may play two competing roles during infection: virus neutralization that leads to protection and recovery, or infection-enhancement that may cause severe complications. Progress in vaccine development has been hampered by limited understanding on protective immunity against dengue virus infection. We report the neutralization activity and infection-enhancement activity in individuals with dengue in Malaysia. We show that infection-enhancement activity is present when neutralizing activity is absent or low, and cross-reactive neutralizing activity may be hampered by infection-enhancing activity. Conventional assays for titration of neutralizing antibody do not consider infection-enhancement activity. We used an alternative assay that determines the sum of neutralizing and infection-enhancement activity in sera from dengue patients. In addition to providing insights into antibody responses during infection, the alternative assay provides a new platform for the study of immune responses to vaccine

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection

Congenital Zika Virus Infection in a Birth Cohort in Vietnam, 2017?2018

To detect congenital ZIKV infection (CZI) in a birth cohort and among high-risk neonates in Vietnam, we collected umbilical cord blood plasma samples of newly delivered babies and peripheral plasma samples of high-risk neonates in Nha Trang, central Vietnam, between July 2017 and September 2018. Samples were subjected to serological and molecular tests. Of the 2013 newly delivered babies, 21 (1%) were positive for Zika virus (ZIKV) IgM and 1,599 (79%) for Flavivirus IgG. Among the 21 ZIKV IgM-positives, 11 were confirmed to have CZI because their plasma samples had anti-ZIKV neutralization titers 3 4 times higher than those against dengue virus (DENV)-1 to 4 and Japanese encephalitis virus (JEV) and were tested for the ZIKV RNA positive by real-time reverse transcription-PCR. Therefore,the incidence of CZI in our birth cohort was approximately 0.5%. Of the 150 high-risk neonates, three (2%) and 95 (63%) were positive for ZIKV IgM and Flavivirus IgG antibodies, respectively. None of the three ZIKV IgM-positives had 3 4 times higher anti-ZIKV neutralization titers than those against DENV-1 to 4 and JEV, and were therefore considered as probable CZI. Our results indicate that CZI is not rare in Vietnam. Although those with confirmed CZI did not show apparent symptoms suspected of congenital Zika syndrome at birth, detailed examinations and follow-up studies are needed to clarify the CZI impact in Vietnam. This is the first report of CZI cases in a birth cohort in Asia

The 2017 Dengue virus 1 outbreak in northern Vietnam was caused by a locally circulating virus group

Background: Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV.Objectives: In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR.Study design: In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained.Results: All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses

Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity