The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle.

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. We interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi.

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.

Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship.

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant-microbe functioning, thus facilitating low-input, sustainable agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Chikungunya Fever Outbreak, Zhejiang Province, China, 2017.

We report a disease outbreak caused by chikungunya virus in Zhejiang Province, China, in August 2017. Phylogenic analysis indicated that this virus belonged to the Indian Ocean clade of the East/Central/South African genotype and was imported by a traveler returning from Bangladesh.

Characterization of a large sex determination region in Salix purpurea L. (Salicaceae).

Dioecy has evolved numerous times in plants, but heteromorphic sex chromosomes are apparently rare. Sex determination has been studied in multiple Salix and Populus (Salicaceae) species, and P. trichocarpa has an XY sex determination system on chromosome 19, while S. suchowensis and S. viminalis have a ZW system on chromosome 15. Here we use whole genome sequencing coupled with quantitative trait locus mapping and a genome-wide association study to characterize the genomic composition of the non-recombining portion of the sex determination region. We demonstrate that Salix purpurea also has a ZW system on chromosome 15. The sex determination region has reduced recombination, high structural polymorphism, an abundance of transposable elements, and contains genes that are involved in sex expression in other plants. We also show that chromosome 19 contains sex-associated markers in this S. purpurea assembly, along with other autosomes. This raises the intriguing possibility of a translocation of the sex determination region within the Salicaceae lineage, suggesting a common evolutionary origin of the Populus and Salix sex determination loci.

Development of multiplex real-time reverse-transcriptase polymerase chain reaction assay for simultaneous detection of Zika, dengue, yellow fever, and chikungunya viruses in a single tube.

Zika virus (ZIKV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) share the same mosquito vectors and have similar clinical manifestations early stage of infection. Therefore, simultaneously differentiating these viruses from each other is necessary. We developed a multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the differentiation of these four viruses in a single tube. The linear range was established by regression analysis, and the R2 value for each virus was ≥0.98, and the 95% lower limit of detection for each virus was as follows (copies/reaction): ZIKV-Asian, 9; ZIKV-Africa, 15; CHIKV, 11; DENV-1, 19; DENV-2, 13; DENV-3, 24; DENV-4, 36; and YFV, 17. Meanwhile, our multiplex real-time RT-PCR has a good consistency with the commercial singleplex assay. In summary, the developed assay can be effectively used for the diagnosis of ZIKV, DENV, CHIKV, and YFV infections.

Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.

Genome-scale model development and genomic sequencing of the oleaginous clade Lipomyces.

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, and 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces.

Tracing the spatiotemporal phylodynamics of Japanese encephalitis virus genotype I throughout Asia and the western Pacific.

BACKGROUND: Japanese encephalitis virus (JEV; Flaviridae: Flavivirus) causes Japanese encephalitis (JE), which is the most important arboviral disease in Asia and the western Pacific. Among the five JEV genotypes (GI-V), GI has dominated traditional epidemic regions in the past 20 years. We investigated the transmission dynamics of JEV GI through genetic analyses. METHODS: We generated 18 JEV GI near full length sequences by using multiple sequencing approaches from mosquitoes collected in natural settings or from viral isolates obtained through cell culture. We performed phylogenetic and molecular clock analyses to reconstruct the evolutionary history by integrating our data with 113 publicly available JEV GI sequences. RESULTS: We identified two subtypes of JEV GI (GIa and GIb), with a rate of 5.94 × 10-4 substitutions per site per year (s/s/y). At present, GIa still circulates within a limited region, exhibited no significant growth, the newest strain was discovered in China (Yunnan) in 2017, whereas most JEV strains circulating belong to the GIb clade. During the past 30 years, two large GIb clades have triggered epidemics in eastern Asia: one epidemic occurred in 1992 [95% highest posterior density (HPD) = 1989-1995] and the causative strain circulates mainly in southern China (Yunnan, Shanghai, Guangdong, and Taiwan) (Clade 1); the other epidemic occurred in 1997 (95% HPD = 1994-1999) and the causative strain has increased in circulation in northern and southern China during the past 5 years (Clade 2). An emerging variant of Clade 2 contains two new amino acid markers (NS2a-151V, NS4b-20K) that emerged around 2005; this variant has demonstrated exponential growth in northern China. CONCLUSION: JEV GI stain circulating in Asia have shifted during the past 30 years, spatiotemporal differences were observed among JEV GI subclade. GIa is still circulating within a limited range, exhibite no significant growth. Two large GIb clades have triggered epidemics in eastern Asia, all JEV sequences identified in northern China during the past 5 years were of the new emerging variant of G1b-clade 2.

[Identification of genotype III Japanese encephalitis virus isolated in Zhejiang province].

OBJECTIVE: To investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983. METHODS: A total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software. RESULTS: From the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology. CONCLUSION: The JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.

Revisiting a 'simple' fungal metabolic pathway reveals redundancy, complexity and diversity.

Next to d-glucose, the pentoses l-arabinose and d-xylose are the main monosaccharide components of plant cell wall polysaccharides and are therefore of major importance in biotechnological applications that use plant biomass as a substrate. Pentose catabolism is one of the best-studied pathways of primary metabolism of Aspergillus niger, and an initial outline of this pathway with individual enzymes covering each step of the pathway has been previously established. However, although growth on l-arabinose and/or d-xylose of most pentose catabolic pathway (PCP) single deletion mutants of A. niger has been shown to be negatively affected, it was not abolished, suggesting the involvement of additional enzymes. Detailed analysis of the single deletion mutants of the known A. niger PCP genes led to the identification of additional genes involved in the pathway. These results reveal a high level of complexity and redundancy in this pathway, emphasizing the need for a comprehensive understanding of metabolic pathways before entering metabolic engineering of such pathways for the generation of more efficient fungal cell factories.

CreA-mediated repression of gene expression occurs at low monosaccharide levels during fungal plant biomass conversion in a time and substrate dependent manner.

Carbon catabolite repression enables fungi to utilize the most favourable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. CreA-mediated regulation has mainly been studied at high monosaccharide concentrations, an uncommon situation in most natural biotopes. In nature, many fungi rely on plant biomass as their major carbon source by producing enzymes to degrade plant cell wall polysaccharides into metabolizable sugars. To determine the role of CreA when fungi grow in more natural conditions and in particular with respect to degradation and conversion of plant cell walls, we compared transcriptomes of a creA deletion and reference strain of the ascomycete Aspergillus niger during growth on sugar beet pulp and wheat bran. Transcriptomics, extracellular sugar concentrations and growth profiling of A. niger on a variety of carbon sources, revealed that also under conditions with low concentrations of free monosaccharides, CreA has a major effect on gene expression in a strong time and substrate composition dependent manner. In addition, we compared the CreA regulon from five fungi during their growth on crude plant biomass or cellulose. It showed that CreA commonly regulated genes related to carbon metabolism, sugar transport and plant cell wall degrading enzymes across different species. We therefore conclude that CreA has a crucial role for fungi also in adapting to low sugar concentrations as occurring in their natural biotopes, which is supported by the presence of CreA orthologs in nearly all fungi.

[Diagnosis of a dengue fever outbreak in Yiwu city, Zhejiang province in 2009 and its molecular tracing of the pathogen].

OBJECTIVE: To determine a dengue fever outbreak in Yiwu city, Zhejiang Province in 2009 and to trace the origin of the pathogen. METHODS: The dengue virus IgM, IgG antibodies and viral nucleic acid were detected and virus was isolated using 40 serum samples from the suspected patients. The viral RNA of the isolated virus strains was extracted and the E gene was amplified by RT-PCR. The amplicons were sequenced and the phylogenetic and homological analyses were also constructed. RESULTS: Among 40 serum samples from dengue fever suspected patients, 17 were positive from for dengue IgM (42.5%); 4 were IgG positive (10.0%); 34 samples were dengue virus RNA positive (85.0%), 28 dengue virus type 3 (D3) strains were isolated (70.0%). The complete coding region of envelope genes (E) from 13 D3 strains was all 1479 nt without any insertion or deletion, which encoded with 493 amino acids (aa). E gene from the 13 D3 strains from Zhejiang in 2009 (D3/ZJ/2009) was 100.0% identical. The strain from Saudi Arabia shared the highest similarity with the D3 strain, 99.3% and 100.0% of their E genes and deduced amino acids were identical, respectively, whereas they were 93.4% and 97.4% between D3/ZJ/2009 strain and its prototype strain (D3/H87/1956), and 93.6% and 97.4% between D3/ZJ/2009 and a D3 strain isolated in Guangxi Province in 1980. The phylogenetic tree of E genes also indicated that D3/ZJ/2009 had maximum similarity with the D3/Saudi Arabia/2004. They all belonged to the D3/GIII branch, which was originated from Indian Subcontinent. CONCLUSION: The outbreak of dengue fever in Zhejiang in 2009 was caused by type 3 dengue virus III genotype. The virus was most likely originated from Saudi Arabia.

Serological and molecular epidemiology of Japanese Encephalitis in Zhejiang, China, 2015-2018.

BACKGROUND: Shifts have occurred in the epidemiological characteristics of Japanese encephalitis (JE), extending from the molecular level to the population level. The aim of this study was to investigate the seroprevalence of JE neutralizing antibodies in healthy populations from different age groups in Zhejiang Province, and to conduct mosquito monitoring to evaluate the infection rate of Japanese encephalitis virus (JEV) among vectors, as well as the molecular characteristics of the E gene of isolated JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1190 sera samples were screened by a microseroneutralization test, including 429 infants (28d-11m) and 761 participants (2y-82y). For those under 1 year old, the geometric mean titers (GMTs) of the JE neutralizing antibody was 9.49 at birth and significantly declined as the age of month increased (r = -0.225, P<0.001). For those above 1-year old, seropositive proportions were higher in subjects aged 1-3 years old as well as ≥25 years old (65%-75%), and relatively lower in subjects aged between 4-25 years old (22%-55%). Four or more years after the 2nd dose of JEV-L (first dose administered at 8 months and the second at 2 years of age), the seropositive proportion decreased to 32.5%, and GMTs decreased to 8.08. A total of 87,201 mosquitoes were collected from livestock sheds in 6 surveillance sites during 2015-2018, from which 139 E gene sequences were successfully amplified. The annual infection rate according to bias-corrected maximum likelihood estimation of JEV in Culex tritaeniorhynchus was 1.56, 2.36, 5.65 and 1.77 per 1000, respectively. JEV strains isolated during 2015-2018 all belonged to Genotype I. The E gene of amplified 139 samples differed from the JEV-L vaccine strain at fourteen amino acid residues, including the eight key residues related to virulence and virus attenuation. No divergence was observed at the sites related to antigenicity. CONCLUSIONS/SIGNIFICANCE: Zhejiang Province was at a high risk of JE exposure due to relatively lower neutralizing antibody levels among the younger-aged population and higher infection rates of JEV in mosquitoes. Continuous, timely and full coverage of JE vaccination are essential, as well as the separation of human living areas and livestock shed areas. In addition, annual mosquito surveillance and periodic antibody level monitoring are important for providing evidence for improvement in JE vaccines and immunization schedules.

Acute Flaccid Myelitis in Children in Zhejiang Province, China.

In July-December 2018, an outbreak of polio-like acute flaccid myelitis (AFM) occurred in Zhejiang province, China. Enterovirus (EV)-D68 infection has been reported to be associated with AFM. This study aimed to investigate the clinical presentation, laboratory findings, and outcomes of AFM patients. We investigated the clinical and virologic information regarding the AFM patients, and real-time PCR, sequencing, and phylogenetic analysis were used to investigate the cause of AFM. Eighteen cases met the definition of AFM, with a median age of 4.05 years (range, 0.9-9 years), and nine (50%) were EV-D68 positive. Symptoms included acute flaccid limb weakness and cranial nerve dysfunction. On magnetic resonance imaging, 11 (61.1%) patients had spinal gray matter abnormalities. Electromyography results of 16 out of 17 patients (94.1%) were abnormal. Cerebrospinal fluid (CSF) pleocytosis was common (94.4%), while CSF protein concentration was normal in all patients. There was little improvement after early aggressive therapy. Phylogenetic analysis revealed that EV-D68 subclade B3 was the predominant lineage circulating in Zhejiang province in 2018.

[Comparative analysis of measles genome between vaccine strain and wild-type strain in Zhejiang province of China].

OBJECTIVE: To explore the distinction between wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level. METHODS: After sequencing of measles wild-stain MVi/Zhejiang. CHN/7.05/4, the distinction between the wild-type strain and the vaccine strain was analysed by MEGA 3.1 software at genome level, and the antigen variation was studied by means of combining the epidemiological data. RESULTS: There were 822 nucleotide differences (5.17%) and 161 amino acid differences between these two strains, including three glycosylation sites variation found. Meanwhile, the antigen ratio between wild-type strain and vaccine strain was found to be 5.66. CONCLUSION: There should be certain differences between the contemporary wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level, and the protective effects of measles vaccine should be studied further.

Sero-molecular epidemiology of enterovirus-associated encephalitis in Zhejiang Province, China, from 2014 to 2017.

BACKGROUND: Recently, both sporadic and outbreak aseptic meningitis caused by enteroviruses have been reported in Zhejiang Province based on a surveillance system. METHODS: This study analysed the epidemiologic features, phylogenetic characteristics and prevalence of enterovirus neutralizing antibodies (nAbs) from 2014 to 2017 in Zhejiang Province. RESULTS: A total of 584 samples were collected. Males accounted for 66.07% while females accounted for 33.93%. The median age was 6 years (range: 1-15 years). Cases peaked in May and August (81.17%) and 162 cases (28.93%) occurred in June. We detected 15 serotypes, some of which (E6, E9, E18 and E30) were the dominant serotypes prevalent in different years and geographical regions. Phylogenetic results revealed that all of the isolates from this study belonged to the human enterovirus B family. A total of 329 subjects sampled from a healthy population were tested for nAbs against B5, E6 and E30 in Rui'an county in 2015. The seropositive rate of E30 in each age group was significantly higher than that of the other serotypes. CONCLUSION: Enterovirus-associated encephalitis pathogens circulating in Zhejiang caused sporadic aseptic meningitis in children. The level of nAbs against human enterovirus reflects the history of previous infections in different age groups. Therefore, additional surveillance sites and more precise seroprevalence studies based on these populations are required to gain better insight into the epidemiology of enterovirus-associated encephalitis in Zhejiang Province.