Enterovirus D68 in Viet Nam (2009-2015)

Background: Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015. Methods: Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam 2009 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively. Results: EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 – 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008. Conclusions: We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis

Clinical, etiological and epidemiological investigations of hand, foot and mouth disease in southern Vietnam during 2015 - 2018

Hand, foot and mouth disease (HFMD) continues to challenge Asia with pandemic potential. In Vietnam, there have been two major outbreaks occurring during 2011-2012 (>200,000 hospitalizations and >200 deaths) and more recently in 2018 (>130,000 hospitalizations and 17 deaths). Given the high burden and the complex epidemic dynamics of HFMD, synthesizing its clinical and epidemiological data remains essential to inform the development of appropriate interventions and design public health measures. We report the results of a hospital-based study conducted during 2015-2018, covering the severe HFMD outbreak recently documented in Vietnam in 2018. The study was conducted at three major hospitals responsible for receiving HFMD patients from southern Vietnam with a population of over 40 million. A total of 19 enterovirus serotypes were detected in 1196 HFMD patients enrolled in the clinical study during 2015-2018, with enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), CV-A10 and CV-A16 being the major causes. Despite the emergence of coxsackieviruses, EV-A71 remains the leading cause of severe HFMD in Vietnam. EV-A71 was consistently detected at a higher frequency during the second half of the years. The emergence of EV-A71 subgenogroup C4 in late 2018 was preceded by its low activity during 2017-early 2018. Compared with EV-A71 subgenogroup B5, C4 was more likely to be associated with severe HFMD, representing the first report demonstrating the difference in clinical severity between subgenogroup C4 and B5, the two predominant EV-A71 subgenogroups causing HFMD worldwide. Our data have provided significant insights into important aspects of HFMD over four years (2015-2018) in Vietnam, and emphasize active surveillance for pathogen circulation remains essential to inform the local public health authorities in the development of appropriate intervention strategies to reduce the burden of this emerging infections. Multivalent vaccines are urgently needed to control HFMD

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples

Enterovirus A71 phenotypes causing hand, foot and mouth disease, Vietnam

We investigated enterovirus A71-associated hand, foot and mouth disease in Vietnam and found that, after replacing subgenogroup C4 in 2013, B5 remained the leading cause of this disease. In contrast with previous observations, this switch did not result in an explosive outbreak, and B5 evolution was driven by negative selection

Enterovirus A71 phenotypes causing hand, foot and mouth disease, Vietnam

We investigated enterovirus A71-associated hand, foot and mouth disease in Vietnam and found that, after replacing subgenogroup C4 in 2013, B5 remained the leading cause of this disease. In contrast with previous observations, this switch did not result in an explosive outbreak, and B5 evolution was driven by negative selection

Emerging coxsackievirus A6 causing hand, foot and mouth disease, Vietnam

Hand, foot and mouth disease (HFMD) is a major public health issue in Asia and has global pandemic potential. Coxsackievirus A6 (CV-A6) was detected in 514/2,230 (23%) of HFMD patients admitted to 3 major hospitals in southern Vietnam during 2011–2015. Of these patients, 93 (18%) had severe HFMD. Phylogenetic analysis of 98 genome sequences revealed they belonged to cluster A and had been circulating in Vietnam for 2 years before emergence. CV-A6 movement among localities within Vietnam occurred frequently, whereas viral movement across international borders appeared rare. Skyline plots identified fluctuations in the relative genetic diversity of CV-A6 corresponding to large CV-A6–associated HFMD outbreaks worldwide. These data show that CV-A6 is an emerging pathogen and emphasize the necessity of active surveillance and understanding the mechanisms that shape the pathogen evolution and emergence, which is essential for development and implementation of intervention strategies

Enterovirus D68 in Viet Nam (2009-2015)

Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, but especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015.Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam between 2009 and 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively.EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 - 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008.We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis