Cooperative effects of three preservatives on physiological quality, endophytic bacterial community and volatile organic compounds of postharvest Codonopsis pilosula var. modesta roots.

BACKGROUND: Codonopsis pilosula var. modesta (CPVM) is a famous medicinal and edible plant of Campanulaceae. However, fresh CPVM roots (FCPVR) are prone to softening, browning and spoilage after concentrated harvesting in the main production area of Gansu Province, China in autumn, which poses great challenges to their large-scale storage and modern processing. In this study, effects of chitosan (CS), natamycin (NA) and modified atmosphere agent (MA) on the postharvest quality of FCPVR were first investigated. The roots after different treatments were stored at 4 °C and relative humidity of 75 ± 5% for 100 days. Their overall quality changes were evaluated from three perspectives: physiological quality, endophytic bacterial community and volatile organic compounds. RESULTS: The clustering heatmap and principal component analysis results indicated that CS (2 g kg-1), NA (0.5 g kg-1) and MA (5 g) had a synergistic effect on physiological quality. The roots in the CS + NA + MA group maintained better physiological state, effective components and antioxidant capacity throughout the storage process. On this basis, compared with room temperature storage, the relative abundance of the main spoilage bacterium Pseudomonas in the CS + NA + MA group roots decreased by 44% on the 100th day of storage. Furthermore, after CS + NA + MA composite treatment, the roots produced richer esters with fruit aroma during low-temperature storage. CONCLUSIONS: The CS + NA + MA composite treatment could maintain the physiological quality and flavor of FCPVR, inhibit spoilage by microbial contamination and maintain the optimal quality during low-temperature storage for up to 100 days. © 2024 Society of Chemical Industry.

Metagenomic sequencing reveals viral diversity of mosquitoes from Shandong Province, China.

Mosquitoes carry a large number of known and unknown viruses, some of which could cause serious diseases in humans or animals. Metagenomic sequencing for mosquito viromes is crucial for understanding the evolutionary history of viruses and preventing emerging mosquito-borne diseases. We collected 1,598 mosquitoes belonging to four species from five counties in Shandong Province, China in 2021. They were grouped by species and sampling locations and subjected to metagenomic next-generation sequencing for the analysis of the viromes. A total of 233,317,352 sequencing reads were classified into 30 viral families and an unclassified group. Comparative analysis showed that mosquitoes in Shandong Province generally possessed host-specific virome. We detected mosquito-borne viruses including Japanese encephalitis virus, Getah virus, and Kadipiro virus in Culex tritaeniorhynchus and Anopheles sinensis samples. Phylogenetic analysis showed that these pathogenic viruses may have existed in mosquitoes in Shandong Province for a long time. Meanwhile, we identified 22 novel viruses belonging to seven families and the genus Negevirus. Our study comprehensively described the viromes of several common mosquito species in Shandong Province, China, and demonstrated the major role of host species in shaping mosquito viromes. Furthermore, the metagenomic data provided valuable epidemiological information on multiple mosquito-borne viruses, highlighting the potential risk of infection transmission. IMPORTANCE: Mosquitoes are known as the source of various pathogens for humans and animals. Culex tritaeniorhynchus, Armigeres subalbatus, and Anopheles sinensis have been found to transmit the Getah virus, which has recently caused increasing infections in China. Cx. tritaeniorhynchus and Culex pipiens are the main vectors of Japanese encephalitis virus and have caused epidemics of Japanese encephalitis in China in past decades. These mosquitoes are widely present in Shandong Province, China, leading to a great threat to public health and the breeding industry. This study provided a comprehensive insight into the viromes of several common mosquito species in Shandong Province, China. The metagenomic sequencing data revealed the risks of multiple pathogenic mosquito-borne viruses, including Japanese encephalitis virus, Getah virus, and Kadipiro virus, which are of great importance for preventing emerging viral epidemics.

Environmental Surveillance of Human Astroviruses in Jinan City of China, 2020-2021.

Human astroviruses (HAstVs) are a significant etiological agent of acute gastroenteritis in children. In order to investigate the circulation of HAstVs during the COVID-19 pandemic, a 2-year environmental surveillance was conducted in Jinan between 2020 and 2021. A total of 24 sewage samples were collected and concentrated. Real-time PCR indicated a positive rate of 83.3%, 79.2% (19/24), and 62.5% for classic, MLB, and VA types of HAstV in sewage samples, respectively, with genomic copies ranging from 6.4 × 103 to 3.7 × 107, 3.2 × 104 to 2.2 × 106, and 1.2 × 104 to 1.6 × 107 l-1. Next-generation sequencing (NGS) analysis on complete ORF2 amplicons from each sewage concentrate revealed the presence of 11 HAstV types, including HAstV-1, -2, -4, -5, MLB1, and VA1 to VA6, as well as non-human animal astroviruses. The most abundant HAstV types were HAstV-1, -4, and -5, which accounted for 70.3%, 12.6%, and 9.1% of total HAstV reads, respectively. Phylogenetic analysis revealed that the sequences obtained in this study were segregated into multiple transmission lineages, yet exhibited less genetic divergence among themselves than with foreign strains. These findings provide insight into the genotype diversity and genetic characterization of HAstVs during the COVID-19 pandemic, and highlight the effectiveness of utilizing NGS approaches to investigate sewage HAstVs.

Genomic characterization of an Omono River virus isolated from Culex tritaeniorhynchus in eastern China.

Omono River virus (OMRV) is a newly reported, unclassified RNA virus in the family Totiviridae, which infects mosquitoes and bats. In this study, we report the isolation of an OMRV strain SD76 from Culex tritaeniorhynchus captured in Jinan city, China. The cytopathic effect was characterized by cell fusion on C6/36 cell line. Its complete genome was 7611 nucleotides in length, with 71.4-90.4% similarities with other OMRV strains. Phylogenetic analysis based on complete genomes showed all OMRV-like strains can be divided into 3 groups with between-group distances ranging from 0.254 to 0.293. These results revealed that the OMRV isolate had high genetic diversity with those identified previously, and enriched the genetic information of family Totiviridae.

The genetic basis and potential molecular mechanism of yellow-albino northern snakehead (Channa argus).

Body colour is an important economic trait for commercial fishes. Recently, a new colour morph displaying market-favoured yellow skin (termed as yellow-mutant, YM) of northern snakehead (Channa argus) was discovered in China. We confirmed that YM snakehead is an albino with complete loss of melanin in the skin and eyes by histological and ultrastructural observations, and inherited as a recessive Mendelian trait. By applying genomic analysis approaches, in combination with gene knockdown and rescue experiments, we suggested a non-sense mutation in slc45a2 (c.383G > A) is the causation for the YM snakehead. Notably, significantly higher levels of key melanogenesis genes (tyr, tyrp1, dct and pmel) and phospho-MITF protein were detected in YM snakehead than those in wild-type individuals, and the underlying mechanism was further investigated by comparative transcriptomic analysis. Results revealed that differential expressed genes involved in pathways like MAPK, WNT and calcium signalling were significantly induced in YM snakehead, which might account for the increased amount of melanogenesis elements, and presumably be stimulated by fibroblast-derived melanogenic factors in a paracrine manner. Our study clarified the genetic basis of colour variation in C. argus and provided the preliminary clue indicating the potential involvement of fibroblasts in pigmentation in fish.

Detection and diversity of human enteroviruses from domestic sewage in Weishan Lake region, eastern China, 2018-2019.

AIMS: Next generation sequencing (NGS) technology has been widely used in monitoring and identifying microbiomes in sewage. We aimed to evaluate the ability of NGS analysis in directly detecting enterovirus (EV) in sewage and to understand the diversity of EVs circulated in the residents in Weishan Lake region. METHODS AND RESULTS: Fourteen sewage samples were collected in Jining, Shandong Province, China from 2018 to 2019 and were parallelly investigated by the P1 amplicon-based NGS method and cell culture method. The results showed that 20 different serotypes belonging to species Enterovirus A (EV-A) (n = 5), EV-B (n = 13), and EV-C (n = 2) were identified by NGS in the sewage concentrates, which exceeded the number of types detected by cell culture method (n = 9). Echovirus 11 (E11), Coxsackievirus (CV) B5 and CVA9 were the most detected types in those sewage concentrates. Phylogenetic analysis revealed that E11 sequences obtained in this study belonged to genogroup D5 and had close genetic relationship with clinical sequences. CONCLUSIONS: Various EV serotypes circulated in populations near Weishan Lake. The combination of NGS technology into environmental surveillance will greatly contribute to improving our knowledge about EV circulation patterns in the population.

Evolutionary phylogeography reveals novel genotypes of coxsackievirus A24 variant and updates the spatiotemporal dynamics in the population with acute hemorrhagic conjunctivitis.

OBJECTIVES: The coxsackievirus A24 variant (CVA24v) has raised a remarkable concern because of its main etiological role in acute hemorrhagic conjunctivitis. METHODS: We conducted a retrospective study to summarize CVA24v isolated from acute hemorrhagic conjunctivitis outbreaks and acute flaccid paralysis surveillance in Shandong province, China during 1988-2020. Phylogenetic and phylogeographic methods based on the VP1 coding region were used to determine the CVA24v origin, spatiotemporal dynamics, and evolution. Also, the positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. RESULTS: The global CVA24vs were classified into eight genotypes (GⅠ-GⅧ). Here, 12 CVA24v isolates were detected, of which five strains were typed as two novel genotypes (GⅦ and GⅧ) and reported first in the world. The time to the most recent common ancestor of the global CVA24v was estimated around March 1965 and evolved with 5.573 × 10-3 substitutions/site/year. Four residues under positive selection were detected, and residue 146T might be adapted in the CVA24v pandemic. Phylogeographic analysis indicated that China was the main source sink for CVA24v dispersion in a long-lasting global pattern. CONCLUSION: Our study updated the epidemiological characteristics of CVA24v and enabled a better understanding of the molecular mechanism underlying different genotypes. The results provided new insights into the CVA24v origin, spatiotemporal dynamics, and possibly, the determinants of viral tropism and pathogenicity.

Detection of multiple human astroviruses in sewage by next generation sequencing.

Human astrovirus (HAstV) composes of classic HAstV serotypes 1-8 and recently discovered novel HAstV-MLB and HAstV-VA strains. A number of studies have demonstrated that wastewater analysis is an effective approach to understand the prevalence and diversity of enteric viruses in local population. However, a comprehensive analysis of classic and novel HAstVs in sewage is still lacking. In this study, sewage samples were collected monthly from Jinan, China during 2018-2019. Quantification of HAstV genomes was performed by real-time quantitative PCR. Different from previous studies which focused on partial ORF1b or ORF2 gene, complete ORF2 region of HAstV was amplified from sewage concentrates, and amplicons were subjected to next generation sequencing (NGS) and genetic analysis. This methodology allowed detection of 18 astroviruses, of which 7 (HAstV-1, -2, -4, -5, VA1, VA2, and VA3) were detected in all sewage samples. A new strain VA6 mapped to the HMO clade was identified in 20.8% of samples, with 82.4%-83.3% nucleotide identities to the closest strain VA5. The viral load of classic, MLB and VA clades in sewage samples ranged from 3.7 × 104 to 4.6 × 107, 3.4 × 104 to 3.9 × 106, and 3.3 × 104 to 4.1 × 106 copies per liter, respectively. Phylogenetic analysis based on complete ORF2 region reflected local HAstVs within each genotype constituted multiple co-circulating lineages. Existence of several new lineages composed exclusively or predominantly of Chinese sequences was observed as well. These results demonstrate sewage contains astroviruses with considerable high diversities. NGS based environmental surveillance greatly improves the understanding of HAstV circulation and should be encouraged.

Identification of the first C1 subgenotype of enterovirus 71 in the Chinese mainland in a retrospective study.

The C4 sub-genotype of Enterovirus 71 (EV71) has been identified as the most dominant sub-genotype circulating in the Chinese mainland since 1998. The circulation situation of EV71 before 1998 is not well established due to insufficient experimental data. The C1 subgenotype of EV71 has not yet been reported in the Chinese mainland by now. Based on the AFP surveillance system of the mainland of China, this study conducted a retrospective study of AFP cases for 1985-1999: a strain of EV-A71 C1 subgenotype was found. To our knowledge, this strain (SD92-41) is the first C1 sub-genotype reported in the Chinese mainland. This study demonstrates that the C1 gene subtype also appeared in the Chinese mainland, but it is unknown whether it is an imported or a local epidemic strain. With sufficient information known from retrospective studies, the source of the SD92-41 strain will be identified and the prevalence of EV-A71 in the Chinese mainland before 1998 will be clearer.

Comparative study on molecular epidemiology of measles H1 outbreak and sporadic cases in Shandong Province, 2013-2019.

BACKGROUND: Measles caused by measles virus (MeV) is a highly contagious viral disease which has also been associated with complications including pneumonia, myocarditis, encephalitis, and subacute sclerosing panencephalitis. The current study isolated 33 strains belonging to 2 groups, outbreak and sporadic strains, in 13 cities of Shandong province, China from 2013 to 2019. Comparison of genetic characterization among 15 outbreak strains and 18 sporadic strains was performed by analyzing nucleotide sequences of the C-terminal region of N protein gene (N-450). RESULTS: All 33 stains belonged to genotype H1. The outbreak strains and sporadic strains distributed crossly in phylogenetic tree. Sequences alignment revealed some interesting G to A transversion which changed the amino acids on genomic sites 1317, 1422, and 1543. The nucleotide and amino acid similarities among outbreak isolates were 98-100% (0-10 nucleotide variations) and 97.7-100%, respectively; They were 97.3-100% and 96.6-100%, respectively for sporadic isolates. Evolutionary genetics analysis revealed that the mean evolution rates of outbreak and sporadic isolates were 1.26 N 10- 3 and 1.48 N 10- 3 substitutions per site per year separately, which were similar with corresponding data before 2012. Local transmission analysis suggested that there were three transmission chains in this study, two of them originated from Japan. Outbreak cases and sporadic cases emerged alternatively and were reciprocal causation on the transmission chains. CONCLUSIONS: Our study investigated the phylogeny and evolutional genetics of MeV during a 7-year surveillance, and compared epidemic and genetic characteristics of outbreak strains and sporadic strains. These results underscore the importance of evolutionary study alongside with sporadic cases in discovering and tracing possible outbreaks, especially in the stage of measles elimination.

Genomic diversity of human papillomavirus type 6 from patients with condyloma acuminatum in Eastern China.

OBJECTIVE: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts both men and women. However, there is limited data on its genomic characterization in mainland China. The aim of this study was to understand the complete genomic diversity of HPV6 from patients with condyloma acuminatum (CA) and to explore the prevalence of different variant lineages/sublineages in eastern China. METHODS: CA samples were collected in 3 hospitals in Shandong Province, China from January 2020 to March 2021. DNA extraction, PCR amplification, Sanger sequencing and sequence assembly were performed on HPV6-positive samples. The complete genomes obtained in this study were analyzed phylogenetically with global HPV6 sequences in GenBank database using MEGA 11. RESULTS: A total of 55 complete genomic sequences of HPV6 were obtained in this study. They were classified as HPV6 variant lineage A (n = 20), sublineage B1 (n = 34) and sublineage B3 (n = 1) by phylogenetic analysis. Sequence alignment showed E1, E5A, E5B, L1, L2, LCR were relatively highly variable regions for sublineage B1 whereas E1, E5A, L2 for lineage A. Both phylogenetic trees of lineage A and sublineage B1 composed of two main branches. Chinese sequences of lineage A segregated into the major branch while those in sublineage B1 belonged to both branches. Genomic divergence between sequences from China and other countries was 0.00% - 0.33% in lineage A and 0.00% - 0.40% in sublineage B1. CONCLUSIONS: This is the first study on HPV variant lineages circulating in mainland China. The results revealed that lineage A and sublineage B1 were prevalent and they had different highly variable regions. Further surveillance is needed to understand the dynamic change of different variants in the population.

Detection of Human Sapoviruses in Sewage in China by Next Generation Sequencing.

Human sapovirus (SaV) is an important causative agent of nonbacterial gastroenteritis in humans. However, little is known about its circulation in China. To study the prevalence and diversity of human SaV genotypes circulating in eastern China, a 3-year environmental surveillance combined with next generation sequencing (NGS) technology was conducted. A total of 36 raw sewage samples were collected from January 2017 to December 2019 in Jinan and processed. Thirty-five (97.22%) samples were positive for human SaV genome in quantitative RT-PCR assay; 33 (91.67%) samples were positive in nested RT-PCR assay on partial capsid VP1 sequence and all amplicons were further analyzed separately by NGS. Among those, ten genotypes belonging to the genogroups of GI, GII, GIV, and GV were identified by NGS, including 4 major genotypes (GI.2, GI.1, GV.1 and GI.3) and 6 uncommon genotypes (GII.5, GII.1, GII.NA1, GII.3, GI.6 and GIV.1). A temporal switch of predominant genotype was observed from GI.2 to GI.1 around June 2019. Local and foreign sequences clustered together in some branches according to phylogenetic analysis, indicating frequent transmission of various lineages in different regions of the world. Environmental surveillance provides a comprehensive picture of human SaV in China. NGS-based environmental surveillance improves our knowledge on human SaV circulating in communities greatly and should be encouraged as a sensitive surveillance tool.

Genetic diversity and molecular epidemiological characterization of group A rotaviruses in raw sewage in Jinan by next generation sequencing.

OBJECTIVE: To understand the genetic diversity and molecular epidemiology characteristics of group A Rotavirus (RVA) in domestic sewage through next generation sequencing (NGS), and to explore the feasibility and necessity of NGS method for RVA environmental surveillance. METHODS: In this study, two sewage samples from Jinan each quarter in 2019 were selected for concentration, RNA extraction, and then RT-PCR reaction. The amplified positive products were subjected to NGS. Finally, the results were analyzed for diversity and phylogeny. RESULTS: A total of 9 G-genotypes and 13 P-genotypes were detected. The Simpson diversity indices in autumn and winter were relatively high. Phylogenetic analysis showed that the dominant types G9 and P[8] were closely related to human-derived sequences. CONCLUSIONS: This study proves that environmental surveillance as a means to understand the prevalence of RVA in the population is not only feasible but necessary. NGS based environmental surveillance greatly improves our understanding on RVA genetic diversity, and should be encouraged as a sensitive surveillance tool.

Prevalence and Bayesian Phylogenetics of Enteroviruses Derived From Environmental Surveillance Around Polio Vaccine Switch Period in Shandong Province, China.

We present the results of environmental surveillance for poliovirus (PV) and non-poliovirus (NPEV) around the switch from trivalent to bivalent oral polio-vaccine (OPV) which occurred in China in May 2016. Sewage samples were collected in Jinan and Linyi city from 2015 to 2017. Enterovirus (EV) isolation, VP1 amplification, Sanger sequencing, and phylogenetic analyses were performed. Among105 sewage samples (36 in Jinan and 69 in Linyi), 101 were positive for EV, with 74.3% (78/105) PV-positive samples and 90.5% (95/105) NPEV-positive samples. A total of 893 EV isolates were obtained, including 326 (36.5%) PVs and 567 (63.5%) NPEVs. Echovirus (E) -11 was the most common serotype out of 18 detected NPEV types (120/567), followed by E-3 (75/567) and E-6 (74/567). PV2 vanished and PV3 came to be the ascendant PV type in sewage after May 2016. Eight PV isolates were judged as pre-vaccine-derived poliovirus (pre-VDPV) and no VDPV or wild PV isolates were monitored. Bayesian phylogenetics demonstrated global E-11 originated in 1876 and evolved with the estimated rate of 4.63 × 10-3 nucleotide substitutions per site per year (s/s/y). Multiple circulating clusters that originated at different times were coexisting in Shandong province. The most recently common ancestor of global coxsackievirus B5 could date back to 1867, at the evolutionary rate of 3.95 × 10-3 s/s/y. In conclusion, our study described the changes of PVs and NPEVs around the polio vaccine switch period and provided meaningful global molecular epidemiological data for further studies of EV-related diseases among the population.

Detection of enteroviruses in urban sewage by next generation sequencing and its application in environmental surveillance.

Environmental surveillance has been used successfully in monitoring enterovirus (EV) circulation; however cell culture method may introduce a selective bias in those EV strains that are recovered from the environment. In this study, urban sewage samples were collected monthly in Jinan, China in 2018 and concentrated via membrane adsorption/elution method. A P1 seminested RT-PCR (RT-snPCR) and NGS method was developed, by which amplicons of 4000 nucleotide in length covering the entire P1 region of EVs were obtained from sewage concentrates and were further analyzed by next generation sequencing (NGS). In addition, for each sewage concentrate, two other assays - cell culture and NGS based partial VP1 amplicon sequencing - were conducted in parallel and compared. The results showed that the P1 RT-snPCR and NGS method generated the most data, with 32 serotypes identified belonging to species EV-A (n = 11), EV-B (n = 14), and EV-C (n = 7). These serotypes covered all those detected from the methods of cell culture (n = 10) and partial VP1 amplicon sequencing (n = 16). EV serotypes from acute flaccid paralysis surveillance correlated with those from sewage. Phylogenetic analysis on coxsackievirus B5, a common pathogen of meningitis, revealed close genetic relationship between environmental and clinical sequences. These results demonstrate sewage contains different EVs related to a variety of diseases. Traditional cell culture method underestimates the existence of some serotypes. NGS based environmental surveillance provides data which are consistent with those from clinical diseases, greatly improves our understanding on the actual circulation in the population, and should be encouraged for public health surveillance.

Environmental Surveillance Complements Case-Based Surveillance of Acute Flaccid Paralysis in Polio Endgame Strategy 2019-2023.

The Polio Endgame Strategy 2019-2023 has been developed. However, more effective and efficient surveillance activities should be conducted with the preparedness of emergence for vaccine-derived poliovirus (VDPV) or wild poliovirus (WPV). We reviewed the impact of the case-based acute flaccid paralysis (AFP) surveillance (1991 to 2018) and environmental surveillance (2011 to 2018) in polio eradication in Shandong province of China. Clinical characteristics of AFP cases and enterovirus (EV) investigation of research samples were assessed. During the period, 10,224 AFP cases were investigated, and 352 sewage samples were collected. The nonpolio AFP rate sustained at over 2.0/100,000 since 1997. Of 10,224 cases, males and young children experienced a higher risk of severe diseases, and 68.5% suffered lower limb paralysis. We collected 1,707 EVs from AFP cases, including 763 polioviruses and 944 nonpolio enteroviruses (NPEVs). No WPV was isolated since 1992. The AFP surveillance showed high sensitivity in detecting 143 vaccine-associated paralytic poliomyelitis (VAPP) cases and 6 VDPVs. For environmental surveillance, 217 (61.6%) samples were positive for poliovirus, and altogether, 838 polioviruses and 2,988 NPEVs were isolated. No WPV was isolated in environmental surveillance, although one VDPV2 was identified. Phylogenetic analysis revealed environmental surveillance had the capacity to detect a large scope of NPEVs. The case-based AFP surveillance will be indispensable for detecting VAPP cases and VDPV circulation in countries using oral polio vaccine. Environmental surveillance is advantageous in identifying EV circulation and responding to ongoing circulating VDPV outbreaks and should be expanded to complement the AFP surveillance.IMPORTANCE Interrupting wild poliovirus transmission and stopping circulating vaccine-derived poliovirus (cVDPV) outbreaks have been proposed as two global goals by the World Health Organization in the Global Polio Eradication Initiative (GPEI). This analysis, based on the 28-year acute flaccid paralysis (AFP) surveillance and 8-year environmental surveillance, provides continued high-quality surveillance performance in achieving the GPEI and detecting the circulation of enterovirus. Given the ongoing cVDPV outbreaks in the world, we present the surveillance capacity of environmental surveillance in capturing enterovirus circulation. The final poliovirus (especially VDPV) elimination has become increasingly complex, and the case-based AFP surveillance alone will lead to difficulties in early detecting dynamics of poliovirus transmission and monitoring the extent of environmental circulation. This study goes beyond previous work to provide a detailed comprehensive evaluation of the enterovirus surveillance and can be used to formulate a set of implementation plan and performance indicators for environmental surveillance.

Complete genome characterization of three enterovirus C96 isolates in China.

Enterovirus C96 (EV-C96) is a newer member of the species Enterovirus C. In this study, we determined the complete genome sequences of three EV-C96 isolates, one recovered from domestic sewage in 2013 and the other two isolated during surveillance of acute flaccid paralysis cases in 1991 and 2009, respectively. The complete genome sequences of these isolates were 75.6-84.2% identical to each other, 75.1-81.8% identical to the prototype strain, and 75.0-91.5% identical to other previously reported strains. Phylogenetic analysis of VP1 sequences revealed a high degree of genetic divergence among currently available EV-C96 sequences in the GenBank database, with an overall mean p-distance of 0.176. It is interesting to note that the 1991 strain 127/SD/CHN/1991 is the earliest EV-C96 isolate so far. Although EV-C96 is not frequently isolated during enterovirus surveillance, its great genetic diversity and the above findings suggest that this serotype has been circulating in China for many years.

Novel astrovirus types circulating in Shandong Province (Eastern China) during 2016: A clinical and environmental surveillance.

BACKGROUND: In recent years, several novel astroviruses have been discovered by molecular assays. Their prevalence in many parts of the world remains unclear. OBJECTIVES: To investigate the existence and genetic diversity of novel astroviruses in China. STUDY DESIGN: Stool specimens were collected from patients suffering gastroenteritis from two hospitals in Jinan city in 2016, and were screened for novel astroviruses by real-time RT-PCR assay. Positive samples were subject of ORF2-amplification by conventional RT-PCR and Sanger sequencing. In addition, 1 liter of sewage sample was collected monthly in 2016 and concentrated via the membrane adsorption/elution method. Partial ORF2 amplification, cloning and Sanger sequencing were conducted, and phylogenetic analysis was performed for genotyping all the obtained sequences. RESULTS: 18 of 635 specimens (2.8%) were positive for novel astrovirus detection by real-time RT-PCR assay and were further genotyped by Sanger sequencing as 13 MLB1, 4 MLB2, and 1 VA3. 100% (12/12) of sewage samples were positive for novel astroviruses by conventional RT-PCR. After cloning and sequencing, six known novel astroviruses (MLB1, MLB2, VA1, VA2, VA3, and VA5) were identified. VA2, MLB1, and VA1 were the most common subtypes being detected in 100.0% (12/12), 91.7% (11/12), and 91.7% (11/12) of the samples, respectively. Eighteen sequences that could not be classified into any known subtypes were found. CONCLUSIONS: These results demonstrate the wide circulation and high diversity of novel astroviruses in Jinan, Shandong Province, China, and underline the significance of environmental surveillance combined with population-based surveillance for understanding the molecular epidemiology of enteric viruses.

Identification and genetic analysis of Kadipiro virus isolated in Shandong province, China.

BACKGROUND: Kadipiro virus (KDV) belongs to the Reoviridae family, which consists of segmented, non-enveloped, double-stranded RNA viruses. It has previously been isolated from Culex, Anopheles, Armigeres and Aedes mosquitoes in Indonesia and China. Here, we describe the isolation and characterization of SDKL1625 from Anopheles sinensis mosquitoes in Shandong province, China. METHODS: In this study, we isolated Kadipiro virus in Aedes albopictus C6/36 cell culture and the complete genome sequencing was made by next generation sequencing. RESULTS: We isolated and characterized a Kadipiro virus from Anopheles sinensis mosquitoes in 2016 in Shandong province, China. Nucleotide and amino acid homology analysis of SDKL1625 showed higher levels of sequence identity with QTM27331 (Odonata, China, 2016) than with JKT-7075 (Culex fuscocephalus, Indonesia, 1981). The SDKL1625 has 86-97% amino acid identity with the JKT-7075, 88-99% amino acid identity with the QTM27331. Among the 12 fragments, VP1, VP2, VP4, VP6, VP7, VP9 and VP12 showed high amino acid identity (> 90%) and VP5 showed the lowest identity (86% and 88%). CONCLUSIONS: This is the first identification of KDV from mosquito in China. Virus morphology and genome organization were also determined, which will further enrich our understanding of the molecular biological characteristics of KDV and seadornaviruses.

Analysis of enterovirus types in patients with symptoms of aseptic meningitis in 2014 in Shandong, China.

We reviewed the epidemiological and clinical characteristics of 927 aseptic meningitis patients in Shandong in 2014, and the phylogeny of predominant enterovirus (EV) types causing this disease was analyzed. A total of 209 patients that were positive for EV were identified by both cell culture and a reverse transcription-seminested PCR in cerebrospinal fluid samples. The positive patients were most likely to be children within 15 years of age, had symptoms such as fever, vomiting and nausea (P＜ .05). The 209 EV sequences belonged to 11 types, and coxsackievirus B5, echovirus types 6 and 30 were predominant types. VP1 analysis exhibited multiple lineages were co-circulating. The significance of the study could come from the fact that surveillance is important to monitor the prevalence of EV types in population, which shows enterovirus meningitis maintains an important public health problem in China.