Insight into the Enterovirus A71: A review.

Enterovirus A71 is a major causative pathogen of hand, foot and mouth disease. It has become a global public health threat, and is especially important for infants and young children in the Asian-Pacific countries. The enterovirus A71 is a non-enveloped virus of the Picornaviridae family having a single-stranded positive-sense RNA genome of about 7.4 kb which encodes the structural and nonstructural proteins. Currently there are no US FDA-approved vaccines or antiviral therapy available against enterovirus A71 infection. Although enterovirus A71 vaccines have been licenced in China, clinically approved vaccines for widespread vaccination programs are lacking. Substantial progress has recently been achieved on understanding the structure and function of enterovirus A71 proteins together with information on the viral genetic diversity and geographic distribution. The present review is intended to provide an overview on our current understanding of the molecular biology and epidemiology of enterovirus A71 which will aid the development of vaccines, therapeutics and other control strategies so as to bolster the preparedness for future enterovirus A71 outbreaks.

Identification of the Viral Pathogens in School Children With Acute Otitis Media in Central Java, Indonesia.

Acute otitis media (AOM) is one of the most common infectious diseases in pediatric clinical facilities and has a significant impact on health care. It is a polymicrobial disease and is usually preceded by a viral upper respiratory tract infection. Data on the spectrum of viruses that cause AOM in Indonesia are still limited. This study analyzed nasopharynx (NP) samples collected from 119 school children with AOM in Banyumas Regency, Central Java, Indonesia. Viral RNA was extracted for cDNA synthesis, followed by PCR and sequencing tools for detection of a panel of respiratory viruses using family-level primers for Coronaviridae, Enterovirus, Bocavirus, and Pneumovirinae for bocavirus. In total, 37 out of 119 NP samples (31.1%) tested positive for viruses. Human rhinovirus B was the predominant virus identified (32.4%) followed by rhinovirus C (29.7%), human rhinovirus A (27%), and human bocavirus (5.4%). Rhinovirus are predominant viral pathogens within school children with AOM in Central Java, Indonesia.

Central nervous system infection in a pediatric population in West Java.

Central nervous system (CNS) viral infections are critical causes of morbidity and mortality in children; however, comprehensive data on etiology is lacking in developing countries such as Indonesia. To study the etiology of CNS infections in a pediatric population, 50 children admitted to two hospitals in Bandung, West Java, during 2017-2018 were enrolled in a CNS infection study. Cerebrospinal fluid and serum specimens were tested using molecular, serological, and virus isolation platforms for a number of viral and bacteriological agents. Causal pathogens were identified in 10 out of 50 (20%) and included cytomegalovirus (n = 4), Streptococcus pneumoniae (n = 2), tuberculosis (n = 2), Salmonella serotype Typhi (n = 1) and dengue virus (n = 1). Our study highlights the importance of using a wide range of molecular and serological detection methods to identify CNS pathogens, as well as the challenges of establishing the etiology of CNS infections in pediatric populations of countries with limited laboratory capacity.

Knowledge, attitudes, and practices associated with zoonotic disease transmission risk in North Sulawesi, Indonesia.

BACKGROUND: Hunters, vendors, and consumers are key actors in the wildlife trade value chain in North Sulawesi, Indonesia, and potentially face an elevated risk of exposure to zoonotic diseases. Understanding the knowledge, attitudes, and practices (KAP) associated with the risk of zoonotic disease transmission in these communities is therefore critical for developing recommendations to prevent or mitigate zoonotic outbreaks in the future. METHODS: Qualitative and quantitative methods were combined to understand KAP associated zoonotic diseases transmission risk in communities involved in the wildlife trade in North Sulawesi. Qualitative data were collected through semi-structured ethnographic interviews and focus group discussions (FGDs) while quantitative data were collected using questionnaires. We conducted 46 ethnographic interviews and 2 FGDs in 2016, and 477 questionnaire administrations in 2017-2018 in communities from five districts in North Sulawesi. We also collected biological specimens, including nasal swab, oropharyngeal swab, and blood, from 254 participants. The study sites were targeted based on known wildlife consumption and trade activities. The participants for qualitative data collection were purposively selected while participants for quantitative data collection were randomly selected. Biological samples were tested for five viral families including Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae and Paramyxoviridae. RESULTS: Knowledge regarding disease transmission from animals to humans was similar across the participants in qualitative focus groups, including knowledge of rabies and bird flu as zoonotic diseases. However, only a small fraction of the participants from the quantitative group (1%) considered that contact with wild animals could cause sickness. Our biological specimen testing identified a single individual (1/254, 0.004%) who was sampled in 2018 with serological evidence of sarbecovirus exposure. Overall, participants were aware of some level of risk in working with open wounds while slaughtering or butchering an animal (71%) but most did not know what the specific risks were. However, significant differences in the attitudes or beliefs around zoonotic disease risk and health seeking behaviors were observed across our study sites in North Sulawesi. CONCLUSIONS: Our study showed variable levels of knowledge, attitudes, and practices associated with the risk of zoonotic disease transmission among study participants. These findings can be used to develop locally responsive recommendations to mitigate zoonotic disease transmission.

Molecular biology of coronaviruses: current knowledge.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) late December 2019 in Wuhan, China, marked the third introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The constant spillover of coronaviruses from natural hosts to humans has been linked to human activities and other factors. The seriousness of this infection and the lack of effective, licensed countermeasures clearly underscore the need of more detailed and comprehensive understanding of coronavirus molecular biology. Coronaviruses are large, enveloped viruses with a positive sense single-stranded RNA genome. Currently, coronaviruses are recognized as one of the most rapidly evolving viruses due to their high genomic nucleotide substitution rates and recombination. At the molecular level, the coronaviruses employ complex strategies to successfully accomplish genome expression, virus particle assembly and virion progeny release. As the health threats from coronaviruses are constant and long-term, understanding the molecular biology of coronaviruses and controlling their spread has significant implications for global health and economic stability. This review is intended to provide an overview of our current basic knowledge of the molecular biology of coronaviruses, which is important as basic knowledge for the development of coronavirus countermeasures.

Pathogenic viruses: Molecular detection and characterization.

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Identification of viral etiology of acute respiratory tract infections in children and adults in Tabanan, Bali, Indonesia.

Acute respiratory tract infection (ARTI) is the most common infectious disease in humans worldwide. The morbidity and mortality rates are high, especially in developing countries from Southeast Asia and Africa. While ARTI is commonly associated with viruses, there is limited data on the spectrum of viruses causing ARTI in developing countries, including Indonesia. This study was based on utilizing molecular techniques targeting a panel of 11 endemic and emerging respiratory viral pathogens including zoonotic viruses in a cohort of children and adults presenting at Tabanan General Hospital, Bali, with acute respiratory illness, from January to November 2017. In total, 98 out of 200 samples (49.0 %) tested positive for viruses. Our study confirmed 64.3 % viral etiology in children and 12.2 % in adults. Viruses that were detected were Herpesviridae (15.0 %) followed by enteroviruses (12.0 %), influenza A virus (11.5 %), respiratory syncytial virus (8.0 %), Adenoviridae (6.5 %), human metapneumovirus (3.5 %), Paramyxoviridae (2.0 %), bocavirus (1.0 %) and Coronaviridae (0.5 %). The study sheds light on the viral spectrum of ARTI in children and adults in Tabanan, Bali, Indonesia.

Detection of multiple viral sequences in the respiratory tract samples of suspected Middle East respiratory syndrome coronavirus patients in Jakarta, Indonesia 2015-2016.

OBJECTIVES: The identification and analysis of viral etiological agents from suspected Middle East respiratory syndrome coronavirus (MERS-CoV) cases admitted to Prof. Dr. Sulianti Saroso Infectious Disease Hospital (IDH) using molecular assays. METHODS: Biological samples were collected from 13 hospitalized patients suspected of MERS-CoV infection in Prof. Dr. Sulianti Saroso IDH from July 2015 to December 2016. The majority of patients presented with pneumonia, with symptoms including fever (≥37.5 °C), labored breathing, and cough, and with a history of travel to the Middle East. Viral RNA was isolated and converted to cDNA, which was used as a template for the detection of 12 viral panels using conventional PCR and sequencing. RESULTS: Viral etiological agents detected in the patients were enterovirus D68, dengue virus type 3, rhinovirus C, human coronavirus 229E, herpes simplex virus type 1, influenza virus H1N1, influenza virus H3N2, human metapneumovirus, and rhinovirus A60. CONCLUSIONS: The sequences of nine viral agents under different taxa were detected in suspected MERS-CoV patients, including influenza virus, paramyxovirus, coronavirus, enterovirus, human metapneumovirus, and herpesvirus.

Birth prevalence and characteristics of congenital cytomegalovirus infection in an urban birth cohort, Jakarta, Indonesia.

OBJECTIVES: Little is known about the birth prevalence and characteristics of congenital cytomegalovirus (CMV) infection in developing countries. To determine the prevalence and characteristics of congenital CMV infection in Indonesia, we conducted a prospective study in an urban birth cohort of neonates at a national referral hospital in 2016-2017, Jakarta, Indonesia. METHODS: Consecutively born neonates were screened for the presence of CMV by using pan-herpesvirus nested-PCR and Sanger sequencing in saliva and/or urine specimens. Both the neonatal clinical findings as well as maternal characteristics were also evaluated. RESULTS: From a total of 411 newborns screened, congenital CMV infection was confirmed in 5.8% of the neonates. These CMV-positive newborns were more likely to have ventriculomegaly and thrombocytopenia compared to CMV-negative neonates. Notably, 67% CMV-positive neonates in our study had clinical findings that required medical intervention, from which only nine presented with symptoms suggestive of congenital CMV infection. Furthermore, congenital CMV infected babies were almost four times more likely to be born to mothers that had placenta previa and placental abruption. CONCLUSIONS: Our work highlights the high prevalence of congenital CMV infection in neonates born in one of the biggest referral hospitals in metropolitan Jakarta, Indonesia.

Characterization of rhinovirus C from a 4-year-old boy with acute onset dilated cardiomyopathy in Jakarta, Indonesia.

INTRODUCTION: Myocarditis, inflammation of the heart muscle, can be caused by infections, autoimmune disease or exposure to toxins. The major cause of myocarditis in the paediatric population is viral infection, including coxsackievirus B3, adenovirus, herpesvirus, parvovirus, influenza A and B, and hepatitis. Here, we report the detection of rhinovirus C in a boy with a clinical presentation of myocarditis, suggesting a possible causative role of this virus in this case. CASE PRESENTATION: A previously well 4.5-year-old boy presented with increasing breathlessness for a week prior to admission. He also had upper respiratory tract infection a few days before the event. An echocardiogram revealed severe left ventricle (LV) systolic dysfunction with dilation of the LV. RNA was extracted from serum and two nasal swabs, and tested with conventional PCR at the family level for viruses including enterovirus, dengue, chikungunya, influenza, herpesvirus, paramyxovirus and coronavirus. Further characterization of the enterovirus group was carried out using PCR with primers targeting the VP4/VP2 gene, followed by sequencing. Molecular tests showed the presence of rhinovirus C genetic material in both serum and swab samples. Phylogenetic analysis of the VP4/VP2 region showed 96-97 % similarity with the closest strain isolated in Ulaanbaatar (Mongolia) and Japan in 2012. CONCLUSION: We report the possible association of rhinovirus C and myocarditis in a child presenting with acute onset of dilated cardiomyopathy.

Detection of central nervous system viral infections in adults in Manado, North Sulawesi, Indonesia.

Central nervous system (CNS) viral infections are important causes of morbidity and mortality worldwide but the systematic survey of patients admitted to hospitals with CNS infections in many countries, including Indonesia, is limited. To obtain more information regarding the causes of CNS infections in Indonesia, this study was performed to detect and identify viral agents associated with CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia. Adult patients admitted to R.D. Kandou General Hospital with presumed CNS infection were enrolled. Cerebrospinal fluid, serum, and throat swab samples were collected and tested using molecular, serological, and virus isolation assays. A confirmed viral etiology was established in three and a probable/possible in 11 out of 74 patients. The most common was herpes simplex virus 1 (7/74, 9.5%), followed by Epstein-Barr virus (2/74, 2.7%), cytomegalovirus (1/74, 1.4%), enterovirus D68 (1/74, 1.4%), rhinovirus A (1/74, 1.4%), dengue virus (1/64, 1.6%), and Japanese encephalitis virus (1/64, 1.6%). There were 20 fatal cases (27.0%) during hospitalization in which eight were associated with viral causes. We identified herpes simplex virus 1 as the most common cause of CNS infection among adults in North Sulawesi with most of the cases remaining undiagnosed. Our study highlights the challenges in establishing the etiology of viral CNS infections and the importance of using a wide range of molecular and serological detection methods to identify CNS viruses.

Occurrence of measles genotype D8 during a 2014 outbreak in Banjarmasin, South Kalimantan, Indonesia.

OBJECTIVES: An outbreak of measles symptoms occurring in children in Banjarmasin, South Kalimantan, Indonesia in 2014 was investigated. METHODS: Nasal swabs were collected from 23 children (median age 41 months) with fever and other symptoms of measles hospitalized in Ulin General Hospital and Islamic Hospital, Banjarmasin, South Kalimantan. Viral RNA was extracted for cDNA synthesis, followed by PCR and sequencing using paramyxovirus family consensus and N-gene primers. RESULTS: Sixteen measles-positive patients (70%) were identified. Fifteen virus strains belonged to genotype D8 and the remaining one strain was confirmed as belonging to genotype D9. CONCLUSION: Measles virus genotype D8 was detected in an outbreak of measles in South Kalimantan, Indonesia, in 2014.

Genomic characterization of Zika virus isolated from Indonesia.

Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies.

Detection and identification of coxsackievirus B3 from sera of an Indonesian patient with undifferentiated febrile illness.

INTRODUCTION: Coxsackievirus B3 (CVB3) virus has been implicated as the causative agent of various outbreaks of clinical disease, including hand, foot, and mouth diseases, aseptic meningitis, acute myocarditis, and inflammatory cardiomyopathy. METHODOLOGY: Two hundred and nine undiagnosed cryopreserved specimens obtained from factory workers in Bandung, Indonesia, who displayed symptoms of acute febrile illness were gathered. Total RNA was isolated from serum and tested by conventional polymerase chain reaction (PCR) using Enterovirus genus-level primers and confirmed by sequencing. Concurrently, the virus was isolated in LLC-MK2 cells. RESULTS: CVB3 virus was identified in an archived specimen from a patient who presented with symptoms of fever, headache, myalgia, and nausea. Sequencing results of the VP1 region from both the clinical sample and tissue culture supernatant showed 97% homology to a CVB3 virus isolate from Taiwan. Virus propagation in LLC-MK2 cell culture exhibited severe cytopathic effects two days post-inoculation. CONCLUSIONS: We report the first case of CVB3 from an undifferentiated febrile illness specimen from Indonesia.