Coxsackieviruses A6 and A16 associated with hand, foot, and mouth disease in Vietnam, 2008-2017: Essential information for rational vaccine design.

Development of multivalent hand, foot, and mouth disease (HFMD) vaccines against enterovirus A71 (EV-A71) and several non-EV-A71 enteroviruses is needed for this life-threatening disease with a huge economic burden in Asia-Pacific countries. Comprehensive studies on the molecular epidemiology and genetic and antigenic characterization of major causative enteroviruses will provide information for rational vaccine design. Compared with molecular studies on EV-A71, that for non-EV-A71 enteroviruses remain few and limited in Vietnam. Therefore, we conducted a 10-year study on the circulation and genetic characterization of coxsackievirus A16 (CV-A16) and CV-A6 isolated from patients with HFMD in Northern Vietnam between 2008 and 2017. Enteroviruses were detected in 2228 of 3212 enrolled patients. Of the 42 serotypes assigned, 28.4% and 22.4% accounted for CV-A6 and CV-A16, being the second and the third dominant serotypes after EV-A71 (31.7%), respectively. The circulation of CV-A16 and CV-A6 showed a wide geographic distribution and distinct periodicity. Phylogenetic analyses revealed that the majority of Vietnamese CV-A6 and CV-A16 strains were located within the largest sub-genotypes or sub-genogroups. These comprised strains isolated from patients with HFMD worldwide during the past decade and the Vietnamese strains have been evolving in a manner similar to the strains circulating worldwide. Amino acid sequences of the putative functional loops on VP1 and other VPs among Vietnamese CV-A6 and CV-A16 isolates were highly conserved. Moreover, the functional loop patterns of VP1 were similar to the dominant patterns found worldwide, except for the T164K substitution on the EF loop in Vietnamese CV-A16. The findings suggest that the development of a universal HFMD vaccine, at least in Vietnam, must target CV-A6 and CV-A16 as two of the three major HFMD-causing serotypes. Vietnamese isolates or their genome sequences can be considered for rational vaccine design.

Genetic characterization of VP1 of coxsackieviruses A2, A4, and A10 associated with hand, foot, and mouth disease in Vietnam in 2012-2017: endemic circulation and emergence of new HFMD-causing lineages.

While conducting sentinel surveillance of hand, foot, and mouth disease (HFMD) in Vietnam, we found a sudden increase in the prevalence of coxsackievirus A10 (CV-A10) in 2016 and CV-A2 and CV-A4 in 2017, the emergence of which has been reported recently to be associated with various clinical manifestations in other countries. However, there have been only a limited number of molecular studies on those serotypes, with none being conducted in Vietnam. Therefore, we sequenced the entire VP1 genes of CV-A10, CV-A4, and CV-A2 strains associated with HFMD in Vietnam between 2012 and 2017. Phylogenetic analysis revealed a trend of endemic circulation of Vietnamese CV-A10, CV-A4, and CV-A2 strains and the emergence of thus-far undescribed HFMD-causing lineages of CV-A4 and CV-A2. The Vietnamese CV-A10 strains belonged to a genotype comprising isolates from patients with HFMD from several other countries; however, most of the Vietnamese strains were grouped into a local lineage. Recently, emerging CV-A4 strains in Vietnam were grouped into a unique lineage within a genotype comprising strains isolated from patients with acute flaccid paralysis from various countries. New substitutions were detected in the putative BC and HI loops in the Vietnamese CV-A4 strains. Except for one strain, Vietnamese CV-A2 isolates were grouped into a unique lineage of a genotype that includes strains from various countries that are associated with other clinical manifestations. Enhanced surveillance is required to monitor their spread and to specify their roles as etiological agents of HFMD or "HFMD-like" diseases, especially for CV-A4 and CV-A2. Further studies including whole-genome sequencing should be conducted to fully understand the evolutionary changes occurring in these newly emerging strains.

Detection of three independently-generated DS-1-like G9P[8] reassortant rotavirus A strains during the G9P[8] dominance in Vietnam, 2016-2018.

A rapid increase and dominance of G9P[8] Rotavirus A strains occurred in northern Vietnam between 2016 and 2018, during which period there appeared three G9P[8] strains possessing short RNA patterns. To understand how the first-ever G9P[8] strains possessing short RNA patterns were formed, next generation sequencing technology was used to examine the whole genomes of the three strains, i.e., RVA/Human-wt/VNM/RVN16.1024/2016/G9P[8], RVA/Human-wt/VNM/RVN17.0879/2017/G9P[8], and RVA/Human-wt/VNM/RVN18.0197/2018/G9P[8], and those of seven representative G9P[8] strains possessing long RNA patterns. The VP7 genes of the short and long G9P[8] strains were > 99% identical, indicating that the origin was in the co-circulating, dominant, long G9P[8] strains. On the other hand, the VP4 genes likely derived from recently-emerging G1/G3/G8P[8] strains possessing the DS-1 backbone. At the lineage level, however, the backbone genes of any one strain differed from that of the other two in the VP1, VP3 or NSP4 gene. Moreover, even at the nucleotide sequence level of the backbone genes belonging to the same lineage, the identities between the three strains were lower than those expected for the strains deriving from an immediate, common ancestor. Thus, the three strains were likely formed by independent reassortment events in which the VP7 gene of the currently dominant G9P[8] strains was incorporated into co-circulating G1/G3/G8P[8] strains possessing similar yet distinct DS-1-like backbone genes. The observation that all of the three reassortant G9P[8] strains were detected only once among the prevalent, ordinary G9P[8] strains suggests that acquisition of the DS-1-like backbone genes unlikely provided selective advantage over the parental Wa-like G9P[8] strains.

Whole genome characterization of feline-like G3P[8] reassortant rotavirus A strains bearing the DS-1-like backbone genes detected in Vietnam, 2016.

While conducting rotavirus gastroenteritis surveillance in Vietnam, two G3P[8] rotavirus A specimens possessing an identical short RNA electropherotype were detected. They were RVA/Human-wt/VNM/0232/2016/G3P[8] and RVA/Human-wt/VNM/0248/2016/G3P[8], and recovered from 9 and 23 months old boys, respectively. The patients developed diarrhoea within one-week interval in March 2016 but in places >100 km apart in northern Vietnam. Whole genome sequencing of the two G3P[8] rotavirus A strains revealed that their genomic RNA sequences were identical across the 11 genome segments, suggesting that they derived from a single clone. The backbone gene constellation was I2-R2-C2-M2-A2-N2-T2-E2-H2. The backbone genes and the VP4 gene had a virtually identical nucleotide sequences with identities ranging from 99.2 to 100% to the corresponding genes of RVA/Human-wt/VNM/1149/2014/G8P[8]; the prototype of recently-emerging bovine-like G8P[8] reassortant strains in Vietnam. On the other hand, the VP7 gene was 98.8% identical with that of RVA/Human-wt/CHN/E2451/2011/G3P[9], and they were clustered together in the lineage represented by RVA/Cat-tc/JPN/FRV-1/1986/G3P[9]. The observations led us to hypothesise that one of the bovine-like G8P[8] strains bearing the DS-1-like backbone genes reassorted with a locally circulating FRV-1-like strain to gain the G3 VP7 gene and to emerge as a thus-far undescribed feline-like G3P[8] reassortant strain. The identification of feline-like G3P[8] strains bearing the DS-1-like backbone genes exemplifies the strength and necessity of the whole genome sequencing approach in monitoring, describing and understanding the evolutionary changes that are occurring in emerging strains and their interactions with co-circulating strains.

13C and 15N assimilation and organic matter translocation by the endolithic community in the massive coral Porites lutea.

Corals evolved by establishing symbiotic relationships with various microorganisms (the zooxanthellae, filamentous algae, cyanobacteria, bacteria, archaea, fungi and viruses), forming the 'coral holobiont'. Among them, the endolithic community is the least studied. Its main function was considered to be translocation of photo-assimilates to the coral host, particularly during bleaching. Here, we hypothesize that (i) endolithic algae may show similar primary production rates in healthy or bleached corals by changing their pigment ratios, and therefore that similar production and translocation of organic matter may occur at both conditions and (ii) diazotrophs are components of the endolithic community; therefore, N2 fixation and translocation of organic nitrogen may occur. We tested these hypotheses in incubation of Porites lutea with 13C and 15N tracers to measure primary production and N2 fixation in coral tissues and endoliths. Assimilation of the 13C atom (%) was observed in healthy and bleached corals when the tracer was injected in the endolithic band, showing translocation in both conditions. N2 fixation was found in coral tissues and endolithic communities with translocation of organic nitrogen. Thus, the endolithic community plays an important role in supporting the C and N metabolism of the holobiont, which may be crucial under changing environmental conditions.