Genotyping and traceability analysis of norovirus in Yantai between 2017 and 2019.

To investigate the diversity and evolution of noroviruses in Yantai in recent years, this study focused on the coat protein regions of norovirus-positive samples with nucleic acid detection (cycle threshold) values below 30 between 2017 and 2019. A total of 81 sequences were obtained for genotyping. Initially, a high-throughput sequencing approach was established to perform the whole-genome sequencing of multiple typical diarrheal strains. Using bioinformatics software such as BEAST, recombinant variant analysis was performed for each genotype of the norovirus strains, and genetic evolutionary analysis was conducted for the dominant strain GII.4, as well as the rare variant GII.21. The results showed that there were multiple genotypes such as GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.6, GII.13, GII.17, GII.21, and GIX.1 in the positive samples of norovirus from 2017 to 2019. GII.4 is characterized by diverse genotypes, with new changes in antigenic epitopes occurring during the course of the epidemic. This may have led to the emergence of a new pandemic. This suggests a need to strengthen surveillance. The results of this study suggest that attention should be paid to the predominant genotypes prevalent in neighboring countries and regions, and the safety supervision of imported food should be strengthened to aid in the prevention and control of related viruses.

Pathogenetic characteristics of infectious diarrhea in Yantai City, Shandong Province, 2018-2019.

Background: Analysis of the pathogenic spectrum, epidemiological characteristics and molecular epidemiological features of important pathogens of infectious diarrhea in Yantai City, Shandong Province, 2018-2019, were analyzed to provide a reference basis for the prevention and control of infectious diarrhea. Methods: A total of 1514 stool specimens were collected within 3 days of the onset of diarrhea in secondary or higher hospitals in Yantai from 2018-2019, and all specimens were tested for the presence of seven major viruses and three major bacteria by employing reverse transcription ploymeraer chain reaction (RT-PCR). Population and seasonal analyses were also performed on positive samples for infectious diarrhea. The top two ranked rotavirus and norovirus were focused on genotyping and analysis of geographical distribution. Results: The study showed that the high prevalence of infectious diarrhea in Yantai, Shandong Province, for two consecutive years in 2018 and 2019 was in young children aged 1-5 years, accounting for 48.6% of the total number of cases. Viral diarrhea was distributed throughout the year with no obvious seasonal distribution, while bacterial diarrhea was predominant in summer. Of 1514 stool specimens, the total positive rate of specimens was 43.92% (665/1514). One pathogen was detected in 507 specimens, two pathogens in 107 specimens, and three pathogens in 44 specimens, with mixed infections accounting for 22.71% of positive specimens (151/665). Viral diarrheal pathogens accounted for 93.23% (620/665) of positive samples. The percentages of positive samples for Rotavirus (RV), Norovirus GI, Norovirus GII, Enterovirus universal (EV), Enteroadenovirus (EAdV), Sapovirus (SaV), Astrovirus (Astv), Salmonella (SE), Listeria monocytogenes (LiMo), and Vibrio parahaemolyticus (VP) were 48.57%, 3.61%, 15.34%, and 10.68% of the total positive samples. Conclusions: This study analyzed in detail the composition of infectious diarrhea pathogen spectrum, pathogen alternation pattern, seasonal distribution and population distribution of pathogens in Yantai City, Shandong Province, 2018-2019, to provide a basis for improving relevant local preventive measures and reducing the disease burden.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Rotavirus Infection and Genotyping in Yantai, Shandong Province, 2017-2019.

PURPOSE: Rotavirus (RV) ranked first among infectious diarrhea-causing pathogens in Yantai from 2017 to 2019. This study investigated the seroserotypes of RV in Yantai, Shandong, from 2017 to 2019 to identify the dominant serotypes and explore the epidemic pattern, aiming to effectively reduce the infection rate, better guide vaccination, and help in epidemiological prevention and control. METHODS: A total of 2227 human diarrhea samples were collected from 2017 to 2019 in Yantai. The VP7 (G serotype) and VP4 (P serotype) genes of 467 RV-positive samples were amplified using two-round nested reverse transcription-polymerase chain reaction for G/P genotyping. RESULTS: The genotyping results of RV in Yantai from 2017 to 2019 revealed that G9 was the dominant serotype for all G serotypes, P[8] was the dominant serotype for all P serotypes, and G9P[8] was the dominant serotype for all G/P combinations. G9 serotype accounted for 60.84%, 95.65%, and 83.76% of the total RV samples collected in 2017, 2018, and 2019, respectively. P[8] accounted for 75.52%, 94.69%, and 88.89% of the RV-positive samples collected in 2017, 2018, and 2019, respectively. G9P[8] accounted for 60.84%, 94.69%, and 83.76% of the total RV samples collected in 2017, 2018, and 2019, respectively. Of the total 467 samples from 2017 to 2019, G2P[4] accounted for 3.64% (17/467), G3P[8] for 1.28% (6/467), and G1P[8] for 0.86% (4/467). CONCLUSION: This study revealed the epidemiological characteristics of RV infection and the development pattern of dominant serotypes in Yantai in recent years, guiding the selection of RV vaccines. The prioritization of vaccines containing G9 serotype for infants in Yantai in recent years is recommended.

Detection and Genetic Correlation Analysis of Diarrhea Cases and Norovirus in Oysters in Yantai, China.

Background: This study aimed to assess the correlation between Norovirus (NoV), diarrhea, and raw oysters from the eastern coastal areas of Yantai, Shandong, China. Methods: Marine oysters were selected from the three aquatic markets in Laishan district, Yantai City, in March 2019. Meanwhile, 100 fecal samples were collected from patients with diarrhea from the same areas during the same period. Nucleic acids were extracted from these samples and detected by employing reverse transcription polymerase chain reaction (RT-PCR) for NoV GI/GII. The VP1 gene of the coat protein of NoV was amplified by semi-nested RT-PCR and sequenced. Sequence comparison of VP1 was performed with BioEdit software, and the evolutionary tree was constructed with Mega7.0 software. Results: Of the 151 oysters, 42 (27.8%) were positive for NoV. Among them, 32 (21.2%) were GII-positive, 10 (6.6%) were GI-positive, and one GI VP1 sequence was obtained in the oyster samples. Of 100 fecal samples from patients with diarrhea, 38 were GII-positive and 17 were GI-positive. Totally, 19 GII VP 1 sequences and eight GI VP 1 sequences were obtained. Two G1 VP 1 sequences in two fecal samples showed 98.7% nucleotide sequence identity and 99.1% amino acid sequence identity G1 VP 1 acquired in the oyster sample. Conclusions: The results suggest that oysters may be responsible for the spread of NoV in Yantai, Shandong province, China.

Analysis of the Transmission of SARS-CoV-2 Delta VOC in Yantai, China, August 2021.

Objective: Starting 31 July 2021, a SARS-CoV-2 outbreak occurred in Yantai, Shandong Province. The investigation showed that this outbreak was closely related to the epidemic at Nanjing Lukou Airport. In view of the fact that there were many people involved in this outbreak and these people had a complex activity area, the transmission route cannot be analyzed by simple epidemiological investigation. Here we combined the SARS-COV-2 whole-genome sequencing with epidemiology to determine the epidemic transmission route of Yantai. Methods: Thirteen samples of SARS-CoV-2 outbreak cases from 31 July to 4 August 2021 were collected and identified by fluorescence quantitative PCR, then whole-genome deep sequencing based on NGS was performed, and the data were analyzed and processed by biological software. Results: All sequences were over 29,000 bases in length and all belonged to B.1.617.2, which was the Delta strain. All sequences shared two amino acid deletions and 9 amino acid mutations in Spike protein compared with reference sequence NC_045512.2 (Wuhan virus strain). Compared with the sequence of Lukou Airport Delta strain, the homology was 99.99%. In order to confirm the transmission relationship between patients, we performed a phylogenetic tree analysis. The results showed that patient 1, patient 2, and patient 9 belong to an independent branch, and other patients have a close relationship. Combined with the epidemiological investigation, we speculated that the epidemic of Yantai was transmitted by two routes at the same time. Based on this information, our prevention and control work was carried out in two ways and effectively prevented the further spread of this epidemic.

Aetiological Characteristics of Infectious Diarrhea in Yantai City, Shandong Province, China in 2017.

This study aimed to analyse the pathogenic spectrum and epidemiological characteristics of infectious diarrhea in Yantai City, Shandong Province, China and provide a reference for its prevention and control. A total of 713 stool specimens collected within 3 days of diarrhea onset from January to December 2017 at secondary or higher hospitals in Yantai City were tested for 10 causative pathogens, using real-time polymerase chain reaction (RT-PCR). The top two rotaviruses and norovirus were analysed for typing and geographical distribution. The total positive rate was 46.56% (332/713), and 268 of 713 specimens contained at least one pathogen; 64 had at least two pathogens, accounting for 19.28% of the positive specimens (64/332). The positivity rates of rotavirus (RV), norovirus (NoVs) GI, norovirus (NoVs) GII, enterovirus universal (EV), enteric adenoviruses (EAdV), sapovirus (SaV), astrovirus (Astv), Salmonella (SE), Listeria monocytogenes (LiMo), and Vibrio parahaemolyticus (VP) were 20.06% (143/713), 1.82% (13/713), 12.84% (89/713), 10.66% (76/713), 4.07% (29/713), 0.42% (3/713), 2.38% (17/713), 1.54% (11/713), 1.82% (13/713), and 1.54% (11/713), respectively. Infectious diarrhea showed a high prevalence in young children aged 1-5 years, accounting for 48.6% of the total number of cases. Bacterial diarrhea was predominant in summer, and viral diarrhea was distributed throughout the year, without a significant seasonal pattern. Rotavirus is dominated by G9P, accounting for 81.82%, while norovirus is dominated by the GII type and has diverse characteristics. The aetiology of infectious diarrhea in Yantai is mainly viral, with RV, NoVs, EV, EAdV, and Astv being the most frequent pathogens. Continuous surveillance of infectious diarrhea diseases can help us understand its epidemiological and pathogenic characteristics, thereby taking targeted preventive and control measures in different seasons.

Broadband Nonlinear Optical Response of InSe Nanosheets for the Pulse Generation From 1 to 2 µm.

Few-layered InSe nanosheets were fabricated by the simple liquid-phase exfoliation method. The morphology and crystal structure features of InSe nanosheet sample were characterized comprehensively. The photoluminescence (PL) spectrum indicated that the liquid-phase exfoliated InSe nanosheets contained variously layered nanoflakes, where eight layers nanosheets dominate. In addition, the first-principle simulation was carried out to describe the electron density of states (DOS) and the electronic band structures. Moreover, the few-layered InSe nanosheets performed excellent nonlinear absorption properties in a broad spectral band. As an application, the stable passively Q-switched (PQS) lasers with few-layered InSe nanosheets saturable absorbers (SAs) were realized with the operating wavelengths at 1.06, 1.34, and 1.91 µm. The shortest pulse durations were 599, 520, and 210 ns, respectively. Our results confirmed that the few-layered InSe nanosheets could be an excellent candidate for pulsed lasers in wide spectral bands.

Genetic and antigenic characterization of influenza A(H1N1)pdm09 in Yantai, China, during the 2009-2017 influenza season.

OBJECTIVE: This study was performed to determine the antigenic and genetic characteristics and evaluate potential vaccine efficacy of influenza A (H1N1)pdm09 in Yantai from August 2009 to August 2017. MATERIALS AND METHODS: A total of 10 236 swabs were collected among patients with an influenza-like illness who were admitted to two sentinel surveillance hospitals in Yantai, East China, from August 2009 to August 2017. All specimens were cultured in Madin-Darby canine kidney cells and identified by haemagglutination-inhibition assay. Complete sequences of haemagglutinin (HA) and neuraminidase of 51 influenza A(H1N1)pdm09 strains circulating in Yantai were amplified, sequenced and analysed using molecular and phylogenetic methods. The potential vaccine efficacy was calculated using the p epitope model which measured the antigenic variation based on the changes in the dominant epitope of HA. RESULTS: The results showed that most Yantai strains were grouped into genetic clades 1.7, 6C, 6B.1 and 6B.2. The amino acid substitutions accumulated gradually in HA proteins and considerable genetic variation were observed in circulating A(H1N1)pdm09 viruses during the seven influenza seasons. The V241I, N369K, N386K and K432E mutations which may change the binding pattern and affinity of oseltamivir for neuraminidase were detected in the strains circulating in 2016/2017 and 2017/2018 seasons and the recommended vaccine strains could afford optimal protection against the influenza A/H1N1pdm09. CONCLUSIONS: Although influenza A(H1N1)pdm09 viruses acquired significant genetic variation over the course of seven influenza seasons, the recommended vaccine strains still afforded protection against main circulating strains. Continuous epidemiological and virological surveillance are necessary.

Analysis of clinical features and early warning indicators of death from severe fever with thrombocytopenia syndrome.

OBJECTIVE: To determine the clinical features of confirmed cases of severe fever with thrombocytopenia syndrome (SFTS) and to explore the early warning indicators of death from SFTS. METHODS: A retrospective case-control study was performed at a single medical institution in Yantai. A total of 20 SFTS patients who died (death group) during January 2014 to December 2015 and another 40 age- and sex-matched SFTS patients who survived (survivor group) were identified from the case records. The differences in demographic characteristics, clinical signs and symptoms, and laboratory parameters in the early stage of disease were compared between the two groups. Conditional logistic regression was used to identify the independent risk factors for mortality in SFTS patients. RESULTS: Univariate logistic regression analysis showed that a disturbance of consciousness, pulse-temperature deficit, neurological signs, hemorrhagic manifestations, pulmonary infection, decreased lymphocyte percentage, high lactate dehydrogenase and creatine kinase levels, increased serum creatinine, blood urea nitrogen, and C-reactive protein (CRP), hyponatremia, and prolonged activated partial thromboplastin time (APPT) and prothrombin time were associated with mortality. On multivariate logistic regression analysis, the independent predictors of death were neurological signs (odds ratio (OR) 31.247, 95% confidence interval (CI) 4.813-202.853), hemorrhagic manifestations (OR 20.251, 95% CI 2.056-199.443), disturbance of consciousness (OR 15.359, 95% CI 2.139-110.268), hyponatremia (OR 5.280, 95% CI 1.235-22.575), increased CRP (OR 2.641, 95% CI 1.090-6.396), increased serum creatinine (OR 6.776, 95% CI 1.047-43.840), and prolonged APTT (OR 6.018, 95% CI 1.450-24.975). CONCLUSIONS: Neurological signs, hemorrhagic manifestations, disturbance of consciousness, hyponatremia, prolonged APTT, and increased CRP and serum creatinine are risk factors for death in SFTS.

Epidemiological characterizations, pathogen spectrum and molecular characteristics of Coxsackievirus A16 from patients with HFMD in Yantai, Shandong, China between 2011 and 2015.

This study aimed to investigate the epidemiological characterizations and pathogen spectrum of hand, foot, and mouth disease (HFMD) in Yantai City, Shandong Province, China, during 2011-2015, and to study the nucleotide evolution and amino acid variation of coxsackievirus A16 (CV-A16) epidemic strains that caused HFMD. The HFMD epidemic began to rise in March, and became prevalent from May to August, reached its peak in June, and then declined in September every year, children aged one to 5 years-old had the highest incidence rate whereas the incidence in children under 6 months was very low, and there were more males than females. Enterovirus nucleic acid detection using real-time reverse transcription polymerase chain reaction was performed on 2130 clinical specimens collected from patients with HFMD between 2011 and 2015, and 2012 enterovirus positive samples were detected, including 678 CV-A16, 639 EV-A71, and 695 other enteroviruses. In total, 60 CV-A16 isolates were randomly selected each year for virus isolation, of which 33 CV-A16 strains were randomly selected for further characterization because CV-A16 is the predominant serotype that caused HFMD in Yantai City, and a phylogenetic tree based on the VP1 region was constructed. All 33 CV-A16 strains belonged to the Bla and B1b genotypes, with a nucleotide similarity of 87.9-100% and deduced amino acid similarity of 98.6-100%. Compared with the reference strain Tainan/5079/98 (AF177911), amino acid mutations were identified at positions 11, 23, 25, 31, 99, 145, and 289, where differences were observed among 33 strains, indicating a unique mutation map of CV-A16 in Yantai City. Our findings demonstrate the etiologic characteristics of HFMD, provide supporting evidence for the prevention and control of HFMD, and open a promising avenue for vaccine development against HFMD, by targeting CV-A16.

[Prevalence and homology analysis on human and animals severe fever with thrombocytopenia syndrome virus infection in Yantai of Shandong province].

OBJECTIVE: To learn the prevalence of infection of human and animals severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) in Yantai, Shandong province, and to analyze the pathogenic features of SFTSV as well as its relationship between human and animal hosts. METHODS: From April to November in 2011, 3 576 serum samples were collected from domesticated animals, including sheep, cattle, pigs, dogs, chickens, in Laizhou and Penglai areas where fever with thrombocytopenia syndrome frequently occurred among local residents. Total SFTSV antibodies and virus-specific nucleic acids of the serum were tested by ELISA and Real time RT-PCR, respectively. SFTSV infection on each animal was observed in different months. 2 590 human serum samples were also collected in Laizhou and Penglai areas, with IgG antibodies tested by ELISA. Virus was isolated with Vero cells from the serum which SFTSV viral nucleic acids were positive. S fragments were amplified by RT-PCR and sequenced, with homology analysis conducted on these sequences. RESULTS: The overall positive rate of serum samples from animals on the total SFTSV antibodies was 40.24% (1 439/3 576) while the positive rate for specific nucleic acids was 4.56% (163/3 576). The positive rates for SFTSV antibodies were 62.78%, 52.97%, 45.56%, 28.73%, 1.45% and the positive rates for specific nucleic acids were 5.72%, 4.63%, 3.02%, 5.25% and 3.73%, in sheep, cattle, chickens, dogs, pigs, respectively. The antigens/antibodies for SFTSV in animals changed seasonally. The overall positive rate for SFTSV IgG antibody from 2 590 human samples was 5.41%. Thirteen virus strains were isolated from these serum samples (10 strains from human and 3 strains from animals). The nucleotide homology of 13S fragments' sequences ranged from 95.23% to 100.00% and the nucleotide homology with the isolates from other provinces were between 94.72% and 99.13%. The homology was considered to be high. CONCLUSION: High prevalence of SFTSV infections occurred both in human and domestic animals in Yantai city. The nucleotide sequences of SFTSV were highly homologous among human and domestic animals. The findings suggested that domesticated animals might serve as SFTSV proliferation and the hosts for transmission thus should be attached great importance.

[The coastal areas of Yantai human and swine hepatitis E virus genotyping analysis].

OBJECTIVE: Survey of the coastal city of Yantai, from human and swine hepatitis E virus (HEV) genotype correlation. METHOD: Application of reverse transcription nested polymerase chain reaction (RT-nPCR) method for local acute sporadic hepatitis E patients,normal population of HEV-IgM positive and local pig farm pigs were HEV RNA detection. And HEV RNA positive samples for cloning sequencing and sequence analysis. RESULTS: In 16 patients with acute sporadic hepatitis E in 7 cases of RNA positive stool specimens of HEV; 51 IgM positive sera of normal people in specimens with 1 HEV RNA positive; 34 pig bile specimens with 1 HEV RNA positive. Sequence analysis revealed the region HEV strains and swine strains in the ORF2 region of nucleotide sequence homology is 87%-98.1%. 7 strains of hepatitis E virus genotype in patients and 1 strains of swine hepatitis E virus genotypes are type IV, gene sequence homology between the 87%-98.1%; there were 6 patients and porcine gene sequence homology in 93.9%-98.1% between,for type a subtype; 1 patients and porcine gene sequence homology in 87%, for the type D subtype. Normal population of 1 cases of hepatitis E virus genotype for I type D subtype. Human and porcine HEV ORF2 gene fragment and HEV part I-IV representative strains were compared, and the nucleotide sequence homology were 82.5%-100%, 81.7%-92.9%, 81.4%-93.9%, 84.9%-100%. CONCLUSION: The area population prevalence of HEV in the presence of 2 genotype 3 subtype genes, mainly to IV A, in pigs with popular HEV gene with a high homology; HEV type I in the crowd disperses in the presence of.

[Expression of helicobacter pylori alpA gene in lactococcus lactis and its immunogenicity analysis].

AIM: To express Helicobacter pylori (Hp) alpA gene in a live delivery vehicle of lactococcus lactis (L. lactis) and assay the efficacy of the L. lactis-alpA oral vaccine in recipient mice. METHODS: The alpA gene of Hp was amplified by PCR and cloned into the prokaryotic expression vector pNICE: sec. The recombinant vector pNICE: sec-alpA was transformed into Lactococcus lactis strain NZ9000. Then the engineered strain was induced to express recombinant alpA as shown by SDS-PAGE and Western blot. Female ICR mice (CV Grade) were randomly divided into 4 groups and administrated orally with PBS, L. lactis pNICE: sec, L. lactis pNICE: sec-alpA, and the inactivated Hp, respectively. After immunized seven times, the mice were detected for their alpA-specific IgG and IgA. RESULTS: The alpA gene was obtained and successfully cloned into the vector pNICE: sec. The recombinant alpA protein (56,000) was accumulated in L. lactis after the induction of the nisin, accounting for 9.6% of the total bacterial protein. Western bolt confirmed that the alpA protein could be recognized specifically by the anti-Hp serum. The titer of anti-alpA IgG in the pNICE: sec-alpA group, comparable to that in the inactivated Hp group, was higher than that in the pNICE: sec group. The titer of anti-alpA IgA in the pNICE: sec-alpA group was higher than all other groups (P<0.05). CONCLUSION: The oral administration of the engineered alpA-expressing L. lactis induced protective immunity against Hp. Our study provides a certain experimental basis for the use of L. lactis as an antigen-delivering vehicle for the development of Hp oral vaccines.