An in vitro workflow to create and modify infectious clones using replication cycle reaction.

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods.

Vaccine development for mosquito-borne viral diseases.

Mosquito-borne viral diseases are a group of viral illnesses that are predominantly transmitted by mosquitoes, including viruses from the Togaviridae and Flaviviridae families. In recent years, outbreaks caused by Dengue and Zika viruses from the Flaviviridae family, and Chikungunya virus from the Togaviridae family, have raised significant concerns for public health. However, there are currently no safe and effective vaccines available for these viruses, except for CYD-TDV, which has been licensed for Dengue virus. Efforts to control the transmission of COVID-19, such as home quarantine and travel restrictions, have somewhat limited the spread of mosquito-borne viral diseases. Several vaccine platforms, including inactivated vaccines, viral-vector vaccines, live attenuated vaccines, protein vaccines, and nucleic acid vaccines, are being developed to combat these viruses. This review analyzes the various vaccine platforms against Dengue, Zika, and Chikungunya viruses and provides valuable insights for responding to potential outbreaks.

Computer-Aided vaccine design for selected positive-sense single stranded RNA viruses

Spontaneous mutations and lack of replication fidelity in positive-sense single stranded RNA viruses (+ssRNA virus) result in emergence of genetic variants with diverse viral morphogenesis and surface proteins that affect its antigenicity. This high mutability in +ssRNA viruses has induced antiviral drug resistance and ability to overcome vaccines that subsequently resulted in rapid viral evolution and high mortality rate in human and livestock. Computer aided vaccine design and immunoinformatics play a crucial role in expediting the vaccine production protocols, antibody production and identifying suitable immunogenic regions or epitopes from the genome sequences of the pathogens. T cell and B cell epitopes can be identified in pathogens by immunoinformatics algorithms and methods that enhance the analysis of protective immunity, vaccine safety, immunity modelling and vaccine efficacy. This rapid and cost-effective computational vaccine design promotes development of potential vaccine that could induce immune response in host against rapidly mutating pathogens like +ssRNA viruses. Epitope-based vaccine is a striking concept that has been widely employed in recent years to construct vaccines targeting rapidly mutating +ssRNA viruses. Therefore, the present review provides an overview about the current progress and methodology in computer-aided vaccine design for the most notable +ssRNA viruses namely Hepatitis C virus, Dengue virus, Chikungunya virus and Coronaviruses. This review also highlights the applications of various immunoinformatics tools for vaccine design and for modelling immune response against +ssRNA viruses. © 2023, National Institute of Science Communication and Policy Research. All rights reserved.

Abstracts of the papers presented in the International Conference of Indian Virological Society, VIROCON 2022 on "Emerging and Re-emerging Viral Infections Impacting Humans, Animals, Plants, Fish and Environment"

The proceedings contain 206 papers. The topics discussed include: influenza: experiences from Kashmir;outbreaks of different viral etiologies amidst COVID-19 pandemic;development of a colorimetric isothermal (LAMP) assay for rapid detection of monkeypox virus;circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021-22);evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals;molecular characterization of dengue viruses circulating in Pune district, Maharashtra from 2009-2022;isolation and genomic characterization of cell fusing agent virus from aedes aegypti mosquitoes from Assam, India;structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase;integration of HBV receptor NTCP into hepatoma cell using grnome editing;and hepatitis B virus genome targeting using CRISPR/Cas9based gene editing tool.

Retrospective investigation of the origin and epidemiology of the dengue outbreak in Yunnan, China from 2017 to 2018.

Since 2013, a dengue epidemic has broken out in Yunnan, China and neighboring countries. However, after the COVID-19 pandemic in 2019, the number of dengue cases decreased significantly. In this retrospective study, epidemiological and genetic diversity characterizations of dengue viruses (DENV) isolated in Yunnan between 2017 and 2018 were performed. The results showed that the dengue outbreak in Yunnan from 2017 to 2018 was mainly caused by DENV1 (genotype I and genotype V) and DENV2 (Asia I, Asia II, and Cosmopolitan). Furthermore, correlation analysis indicated a significant positive correlation between the number of imported and local cases (correlation coefficient = 0.936). Multiple sequence alignment and phylogenetic divergence analysis revealed that the local isolates are closely related to the isolates from Myanmar and Laos. Interestingly, recombination analysis found that the DENV1 and DENV2 isolates in this study had widespread intra-serotype recombination. Taken together, the results of the epidemiological investigation imply that the dengue outbreak in Yunnan was primarily due to imported cases. This study provides a new reference for further investigations on the prevalence and molecular epidemiology of DENV in Yunnan, China.

Dengue & COVID-19: A Comparison and the Challenges at Hand.

The COVID-19 pandemic caused by SARS-CoV-2 spread across many countries between 2020 and 2022. The similarities in clinical presentation with other endemic diseases pose a challenge to physicians in effectively diagnosing and treating the infection. Approximately 129 nations have a risk of dengue infection, and more than 100 of those are endemic to dengue. During the COVID-19 pandemic, the number of dengue cases decreased in many countries owing to the isolation measures followed. However, the common clinical presentation between them has led to misdiagnosis. Both COVID-19 and dengue fever cause a surge in pro-inflammatory cytokines and chemokines, thus sharing a common pathophysiology. False positive serological test results also posed difficulty differentiating between COVID-19 and dengue fever. This review aims to compare the clinical features, pathophysiology, and immune response between dengue and COVID-19, to benefit public health management during the pandemic.

Visual and Quantitative Analyses of Virus Genomic Sequences using a Metric-based Algorithm

This work aims to study the virus RNAs using a novel accelerated algorithm to explore any-length repetitive genomic fragments in sequences using Hamming distance between the binary-expressed characters of an RNA and a query pattern. Primary attention is paid to the building and analyzing 1-D distributions (walks) of atg-patterns - codon-starting triplets in genomes. These triplets compose a distributed set called a word scheme of RNA. A complete genome map is built by plotting the mentioned atg-walks, trajectories of separate (a-, c-, g-, and t-symbols) nucleotides, and the lines designating the genomic words. The said map can be additionally equipped by gene's designations making this tool pertinent for multi-scale genomic analyses. The visual examination of atg-walks is followed by calculating statistical parameters of genomic sequences, including estimating walk-geometry deviation of RNAs and fractal properties of word-length distributions. This approach is applied to the SARS CoV-2, MERS CoV, Dengue, and Ebola viruses, whose complete genomic sequences are taken from GenBank and GISAID. The relative stability of these walks for SARS CoV-2 and MERS CoV viruses was found, unlike the Dengue and Ebola distributions that showed an increased deviation of their geometrical and fractal characteristics. The developed approach can be useful in further studying mutations of viruses and building their phylogenic trees. © World Scientific and Engineering Academy and Society. All Rights Reserved.

Traditional herbal medicine in the treatment of patients infected with new coronavirus

The purposes of this review was in the direction of epidemiology, causative agents, symptoms, vaccine research probabilities and COVID-19 infection novel Corona viruses that was investigated in China. The COVID-19 has surrounded viruses along with a practical sensation one stranded RNA genome and a nucleocapsid of helical uniformity. The COVID-19 is an enormous family of viruses that are prevalent in a public and large number of species of animals including hens, camels, bats, cat, and cattle. Human corona viruses can cause gentle disorder identical to a common cough, cold, while others reason more acute disease MERS (Middle East Respiratory Syndrome) and SARS (Severe Acute Respiratory Syndrome). Thus spreading the COVID-19 should be closely investigated to recognize the growth of particularly virulent strains in society at an early stage and to simplify the evolution of enough preventive and therapeutic measurements.Copyright © 2021, Health Biotechnology and Biopharma. All rights reserved.

Changes and correlation of dengue fever epidemic and Aedes albopictus density in Haizhu district, Guangzhou before and after the COVID-19 pandemic

To analyze the population density, seasonal fluctuation of Aedes albopictus in Haizhu District, Guangzhou from 2017 to 2021, so as to provide a scientific basis for the monitoring and prevention and control of mosquito vector density of dengue fever. The data of dengue fever cases and Aedes surveillance data in Haizhu District, Guangzhou from 2017 to 2021 were collected, and the data of 2017-2019 and 2020-2021 were grouped to compare and analyze the characteristics of dengue epidemic and the density fluctuation of Aedes mosquitoes. A total of 517 dengue cases were reported in Haizhu District, Guangzhou from 2017 to 2021, of which only 7 cases were reported from 2020 to 2021, and the peak period of reported cases every year was August to November. Before the COVID-19 pandemic, there was a positive correlation between the number of local cases and the number of imported cases(rs=0.63, P<0.05) and BI(rs=0.73, P<0.05). The peak density of Aedes was from May to October, and the differences of mean BI(X~2=1 143.40,P<0.001), MOI(X~2=188.30,P<0.001), and SSI(X~2=4 499.43,P<0.001)before and after the COVID-19 pandemic were statistically significant. In general, before and after the COVID-19 pandemic, the density of Aedes in high-risk areas was higher than that in low-risk areas. After COVID-19 pandemic, the number of reported cases and the density of Aedes in Haizhu District decreased, but the density of Aedes in the high-risk area was still higher than that in low-risk areas, and a certain risk of outbreak still existed, so the government should continue to take more precise measures to strictly prevent dengue epidemic.

Summary of global surveillance data of infectious diseases in January 2023

In January 2023, a total of 64 infectious diseases were reported globally, affecting 235 countries and regions. Except for influenza, the top five infectious diseases affecting greatest number of countries and regions were COVID-19 (235), monkeypox (110), dengue fever (31), measles (27) and cholera (15). The top five infectious diseases with highest case fatality rates were Nipah virus disease (62.5%), Ebola virus disease (47.0%), Crimean-Congo haemorrhagic fever (37.5%), Lassa fever (15.1%) and West Nile fever (7.6%). The top five infectious diseases with greatest number of deaths were COVID-19, malaria, cholera, measles and dengue fever. The prevalent infectious diseases in Asia were COVID-19, cholera and dengue fever, the prevalent infectious diseases in Africa were COVID-19, cholera, yellow fever, Lassa fever, malaria and monkeypox, the prevalent infectious diseases in America were COVID-19, cholera, monkeypox, dengue fever and chikungunya fever, the prevalent infectious disease in Europe were COVID-19, monkeypox and invasive group A streptococcus infection.

Recombinant Modified Vaccinia Virus Ankara Expressing a Glycosylation Mutant of Dengue Virus NS1 Induces Specific Antibody and T-Cell Responses in Mice.

The four serotypes of dengue virus (DENV1-4) continue to pose a major public health threat. The first licenced dengue vaccine, which expresses the surface proteins of DENV1-4, has performed poorly in immunologically naïve individuals, sensitising them to antibody-enhanced dengue disease. DENV non-structural protein 1 (NS1) can directly induce vascular leakage, the hallmark of severe dengue disease, which is blocked by NS1-specific antibodies, making it an attractive target for vaccine development. However, the intrinsic ability of NS1 to trigger vascular leakage is a potential drawback of its use as a vaccine antigen. Here, we modified DENV2 NS1 by mutating an N-linked glycosylation site associated with NS1-induced endothelial hyperpermeability and used modified vaccinia virus Ankara (MVA) as a vector for its delivery. The resulting construct, rMVA-D2-NS1-N207Q, displayed high genetic stability and drove efficient secretion of NS1-N207Q from infected cells. Secreted NS1-N207Q was composed of dimers and lacked N-linked glycosylation at position 207. Prime-boost immunisation of C57BL/6J mice induced high levels of NS1-specific antibodies binding various conformations of NS1 and elicited NS1-specific CD4+ T-cell responses. Our findings support rMVA-D2-NS1-N207Q as a promising and potentially safer alternative to existing NS1-based vaccine candidates, warranting further pre-clinical testing in a relevant mouse model of DENV infection.

Title- to study the clinical profile of patients infected with dengue in a tertiary care center in COVID pandemic era

Dengue infection has always been a major challenge to the public health and in the absence of specific treatment and availability of effective vaccine, this infection has been able to cause repeated outbreaks in different parts of the world for the past many years. This study was planned to analyse the symptoms and complications of dengue positive patients in the backdrop of Covid -19 pandemic. METHODS- A retrospective observational analysis was done on 87 patients presenting to a tertiary care center in northern India by taking into account of their presenting symptoms, haematological parameters and complications. RESULTS- Out of 87 patients 59 (67%) were males and 28 (32%) were females with maximum (36%) belonging to the age group of 18-30 years. 69% patients were from village areas. Fever, headache were the most common symptoms and thrombocytopenia (99%) was the most common haematological complication followed by liver dysfunction (88%). Respiratory symptoms were seen in 18% and skin manifestations were seen in 12% patients. CONCLUSION- Dengue infection continues to be a major cause of morbidity and mortality in many parts of the world. The early recognition of symptoms and signs is imperative to the successful management of disease. With the existence of Covid 19 infection it becomes more important to carefully observe and differentiate between the two viral illnesses as both can have overlapping symptoms.

Summary of global surveillance data of infectious diseases in December 2022

In December 2022, a total of 68 infectious diseases were reported globally, affecting 235 countries and regions. Except for influenza, the top five infectious diseases affecting greatest number of countries and regions were COVID-19 (235), monkeypox (110), dengue fever (28), measles (27) and cholera (14). The top five infectious diseases with highest case fatality rates were Ebola virus disease (47.0%), Rift Valley fever (44.2%), Crimean-Congo haemorrhagic fever (40.0%), Lassa fever (17.6%) and West Nile fever (7.6%). The top five infectious diseases with greatest number of deaths were COVID-19, malaria, cholera, dengue fever and measles. The prevalent infectious diseases in Asia were COVID-19, cholera and dengue fever, the prevalent infectious diseases in Africa were COVID-19, cholera, yellow fever, Lassa fever, monkeypox, malaria and measles, the prevalent infectious diseases in America were COVID-19, cholera, monkeypox, dengue fever and chikungunya fever, the prevalent infectious disease in Europe were COVID-19, monkeypox and invasive group A streptococcus infection.

Ayurvedic and Other Herbal Remedies for Dengue: An Update

Dengue fever is a flu-like ailment propagated by female mosquitos of the Aedes aegypti species. It is also known as dandaka jwara in Ayurveda. It is most common in the world's subtropical and tropical climate zones. Vomiting, severe headache, nausea, rashes, joint pain, pain behind the eyes, muscle pain, and swollen glands are all common dengue symptoms. If not handled promptly, these symptoms can lead to more severe issues such as exhaustion, blood in the vomit, continuous vomiting, bleeding gums, restlessness, severe abdominal pain, and rapid bleeding. Because there is no specific medication for dengue fever, the disease is treated by eliminating and managing the symptoms. Fortunately, there are a variety of ayurvedic remedies (like Carica papaya L., Cissampelos pareira L., etc.) that can help to tackle the same by strengthening the immune system and controlling hyperthermia. This review article provides a comprehensive overview of dengue virus infections, clinical symptoms, diagnosis, mitigation, and treatments, focusing on ayurvedic and herbal remedies.Copyright © 2022 The Author(s)

Enhancing vaccination of key populations: lessons and actions

Vaccination is effective in preventing the increase of disease, especially emerging infectious diseases (EIDs), and it is particularly important for people in close contact with infected sources and susceptible populations who are at increased risk of getting infectious diseases due to behavior, occupation or health. Despite targeted vaccination guidelines, inadequate vaccination of the key populations fails to receive widespread attention, resulting in a high-risk transition of disease from key populations to general populations. Strengthening the vaccination of the susceptible groups can effectively block the spread of pathogens to general populations, and reduce the consumption of medical resources in universal vaccination, which has significant economic value. In this review, we describe the prevalence of EIDs, analyze the experience and lessons of infectious disease vaccination in key populations through several cases, and further explore the causes for the decline in vaccination rates of key populations. According to the trends of EIDs, a plan to strengthen the vaccination of key populations is proposed to effectively prevent the transition of EIDs from key populations to general populations.

Enhancing Knowledge on Prevention of Dengue Hemorrhagic Fever (DHF) through Focus Group Discussion (FGD) and Training of Trainers (TOT) Approaches during the Pandemic Period at Dupak Health Center, Surabaya

Objective: The COVID-19 pandemic that has occurred over the past 2 years has had a very significant impact on various sectors, especially public health. Indonesia, which is a tropical country, especially has to deal with cases of dengue disease which are still quite high. The government's social distancing policy that was in effect during the COVID-19 pandemic caused the control and prevention of dengue fever to be neglected by the public. Purpose(s): This study aims to increase public understanding of dengue prevention through Focus Group Discussion (FGD) and Training of Trainer (TOT) approaches. Method(s): This research was conducted using 2 approaches, namely FGD and TOT, which were followed by 15 and 30 public workers participants. The FGD and TOT were carried out by discussing and giving lectures to participants, then participants were given a pre-test and post-test question sheets. Result(s): As many as 93.3% of participants already know the causes, methods of prevention, and types of dengue mosquitoes. However, there were still 60% of participants understood the handling of mosquito larvae correctly. After the FGD and TOT were carried out, it was seen that there was an increase in participants' understanding. Conclusion(s): The FGD and TOT approaches can increase public understanding of the causes, prevention, and proper inspection of DHF mosquito larvae.Copyright © 2023, Institute of Medico-legal Publication. All rights reserved.

The Role of NS1 Protein in the Diagnosis of Flavivirus Infections.

Nonstructural protein 1 (NS1) is a glycoprotein among the flavivirus genus. It is found in both membrane-associated and soluble secreted forms, has an essential role in viral replication, and modulates the host immune response. NS1 is secreted from infected cells within hours after viral infection, and thus immunodetection of NS1 can be used for early serum diagnosis of dengue fever infections instead of real-time (RT)-PCR. This method is fast, simple, and affordable, and its availability could provide an easy point-of-care testing solution for developing countries. Early studies show that detecting NS1 in cerebrospinal fluid (CSF) samples is possible and can improve the surveillance of patients with dengue-associated neurological diseases. NS1 can be detected postmortem in tissue specimens. It can also be identified using noninvasive methods in urine, saliva, and dried blood spots, extending the availability and effective detection period. Recently, an enzyme-linked immunosorbent assay (ELISA) assay for detecting antibodies directed against Zika virus NS1 has been developed and used for diagnosing Zika infection. This NS1-based assay was significantly more specific than envelope protein-based assays, suggesting that similar assays might be more specific for other flaviviruses as well. This review summarizes the knowledge on flaviviruses' NS1's potential role in antigen and antibody diagnosis.

A Computational Framework for Determining the Breadth of Antibodies Against Highly Mutable Pathogens

Highly mutable pathogens pose daunting challenges for antibody design. The usual criteria of high potency and specificity are often insufficient to design antibodies that provide long-lasting protection. This is due, in part, to the ability of the pathogen to rapidly acquire mutations that permit them to evade the designed antibodies. To overcome these limitations, design of antibodies with a larger neutralizing breadth can be pursued. Such broadly neutralizing antibodies (bnAbs) should remain targeted to a specific epitope, yet show robustness against pathogen mutability, thereby neutralizing a higher number of antigens. This is particularly important for highly mutable pathogens, like the influenza virus and the human immunodeficiency virus (HIV). The protocol describes a method for computing the "breadth” of a given antibody, an essential aspect of antibody design. © 2023, Springer Science+Business Media, LLC, part of Springer Nature.

SARS-CoV-2 and dengue virus co-infection: Epidemiology, pathogenesis, diagnosis, treatment, and management.

SARS-CoV-2 and dengue virus co-infection cases have been on the rise in dengue-endemic regions as coronavirus disease 2019 (COVID-19) spreads over the world, posing a threat of a co-epidemic. The risk of comorbidity in co-infection cases is greater than that of a single viral infection, which is a cause of concern. Although the pathophysiologies of the two infections are different, the viruses have comparable effects within the body, resulting in identical clinical symptoms in the case of co-infection, which adds to the complexity. Overlapping symptoms and laboratory features make proper differentiation of the infections important. However, specific biomarkers provide precise results that can be utilised to diagnose and treat a co-infection, whether it is simply COVID-19, dengue, or a co-infection. Though their treatment is distinguished, it becomes more complicated in circumstances of co-infection. As a result, regardless of whatever infection the first symptom points to, confirmation diagnosis of both COVID-19 and dengue should be mandatory, particularly in dengue-endemic regions, to prevent health deterioration in individuals treated for a single infection. There is still a scarcity of concise literature on the epidemiology, pathophysiology, diagnosis, therapy, and management of SARS-CoV-2 and dengue virus co-infection. The epidemiology of SARS-CoV-2 and dengue virus co-infection, the mechanism of pathogenesis, and the potential impact on patients are summarised in this review. The possible diagnosis with biomarkers, treatment, and management of the SARS-CoV-2 and dengue viruses are also discussed. This review will shed light on the appropriate diagnosis, treatment, and management of the patients suffering from SARS-CoV-2 and dengue virus co-infection.