Evaluation of the protective efficacy of three novel identified membrane associated proteins of Streptococcus suis serotype 2.

Streptococcus suis is one of the major pathogens of pigs circulating worldwide, and the development of vaccines will help to effectively control streptococcosis in swine. In this study, we evaluated the potential of three membrane associated proteins, histidine kinase (HK), glycosyltransferase family 2 (Gtf-2) and phosphate binding protein (PsbP) of S. suis as subunit vaccines. Bioinformatics analysis shows that protein ABC is highly conserved in S. suis. To verify the protective effects of these proteins in animal models, recombinant protein HK, Gtf-2 and PsbP were used to immunize BALB/c mice separately. The results showed that these proteins immunization in mice can effectively induce strong humoral immune responses, protect mice from cytokine storms caused by S. suis infection, and have a significant protective effect against lethal doses of S. suis infection. Furthermore, antibodies with opsonic activity exist in the recombinant proteins antiserum to assist phagocytic cells in killing S. suis. Overall, these results indicated that these recombinant proteins all elicit good immune protective effect against S. suis infection and can be represent promising candidate antigens for subunit vaccines against S. suis.

Transposon library screening to identify genes with a potential role in Streptococcus suis biofilm formation.

Background: Biofilm formation is considered to be one of reasons for difficulty in the prevention and control of Streptococcus suis. Aims: To explore the potential genes involved in the biofilm formation of S. suis. Methods: Transposon mutagenesis technology was used to screen biofilm-defective strains of S. suis, and the potential genes related to biofilm were identified. Results: A total of 19 genes were identified that were involved in bacterial metabolism, peptidoglycan-binding protein, cell wall synthesis, ABC transporters, and so on. Conclusion: This study constructed 979 transposon mutation libraries of S. suis. A total of 19 gene loci related to the formation of S. suis biofilm were identified, providing a reference for exploring the mechanism of S. suis biofilm formation in the future.

Streptococcus suis is an important pathogen (this is a microorganism that causes, or can cause, disease) that can be transmitted between animals and humans. The ability to form a protective community, called a biofilm, is one of the reasons why we can have difficulty in preventing and treating S. suis infection. The main purpose of this study was to screen potential genes that may determine biofilm formation in S. suis. The results revealed 19 genes that may affect the biofilm formation of S. suis.

Rethinking the control of Streptococcus suis infection: Biofilm formation.

Streptococcus suis is an emerging zoonotic pathogen that is widespread in swine populations. The control of S. suis infection and its associated diseases is a daunting challenge worldwide. Biofilm formation appears to be the main reason for the persistence of S. suis. In this review we gather existing knowledge on S. suis biofilm, describing the role of biofilm formation in S. suis virulence and drug resistance, the regulatory factors of S. suis biofilm formation, and the research progress of inhibiting S. suis biofilm formation, with the aim of providing guidance for future studies related to the field of S. suis biofilms.

New antibacterial targets: Regulation of quorum sensing and secretory systems in zoonotic bacteria.

Quorum sensing (QS) is a communication mechanism that controls bacterial communication and can influence the transcriptional expression of multiple genes through one or more signaling molecules, thereby coordinating the population response of multiple bacterial pathogens. Secretion systems (SS) play an equally important role in bacterial information exchange, relying on the secretory systems to secrete proteins that act as virulence factors to promote adhesion to host cells. Eight highly efficient SS have been described, all of which are involved in the secretion or transfer of virulence factors, and the effector proteins they secrete play a key role in the virulence and pathogenicity of bacteria. It has been shown that many bacterial SS are directly or indirectly regulated by QS and thus influence bacterial virulence and antibiotic resistance. This review describes the relationship between QS and SS of several common zoonotic pathogenic bacteria and outlines the molecular mechanisms of how QS systems regulate SS, to provide a theoretical basis for the study of bacterial pathogenicity and the development of novel antibacterial drugs.

Establishment of Streptococcus suis Biofilm Infection Model In Vivo and Comparative Analysis of Gene Expression Profiles between In Vivo and In Vitro Biofilms.

Streptococcus suis is a zoonotic pathogen that continuously threatens animal husbandry and public health worldwide. Studies have shown that S. suis can cause persistent infection by forming biofilms. In this study, a model of S. suis biofilm-related infection was successfully constructed for the first time by simulating the natural infection of S. suis, and biofilm of S. suis in vivo was successfully observed in the lung tissue of infected pigs by a variety of detection methods. Subsequently, selective capture of transcribed sequences (SCOTS) was used to identify genes expressed by S. suis in vivo biofilms. Sixty-nine genes were captured in in vivo biofilms formed by S. suis for the first time by SCOTS; they were mainly involved in metabolism, cell replication, and division, transport, signal transduction, cell wall, etc. Genes related to S. suis in vitro biofilm formation were also identified by SCOTS and RNA sequencing. Approximately half of the genes captured by SCOTS in the in vivo and in vitro biofilms were found to be different. In summary, our study provides powerful clues for future exploration of the mechanisms of S. suis biofilm formation. IMPORTANCE Streptococcus suis is considered an important zoonotic pathogen, and persistent infection caused by biofilm is currently considered to be the reason why S. suis is difficult to control in swine. However, to date, a model of the biofilm of S. suis in vivo has not been successfully constructed. Here, we successfully detected biofilms of S. suis in vivo in lung tissues of piglets infected with S. suis. Selective capture of transcribed sequences and the transcriptome were used to obtain gene profiles of S. suis in vivo and in vitro biofilms, and the results showed large differences between them. Such data are of importance for future experimental studies exploring the mechanism of biofilm formation by S. suis in vivo.

Bringing Antimicrobial Strategies to a New Level: The Quorum Sensing System as a Target to Control Streptococcus suis.

Streptococcus suis (S. suis) is an important zoonotic pathogen. It mainly uses quorum sensing (QS) to adapt to complex and changeable environments. QS is a universal cell-to-cell communication system that has been widely studied for its physiological functions, including the regulation of bacterial adhesion, virulence, and biofilm formation. Quorum sensing inhibitors (QSIs) are highly effective at interfering with the QS system and bacteria have trouble developing resistance to them. We review the current research status of the S. suis LuxS/AI-2 QS system and QSIs. Studies showed that by inhibiting the formation of AI-2, targeting the LuxS protein, inhibiting the expression of luxs gene can control the LuxS/AI-2 QS system of S. suis. Other potential QSIs targets are summarized, which may be preventing and treating S. suis infections, including AI-2 production, transmission, LuxS protein, blockage of AI-2 binding to receptors, AI-2-mediated QS. Since antibiotics are becoming increasingly ineffective due to the emergence of resistant bacteria, including S. suis, it is thus critical to find new antibacterial drugs with different mechanisms of action. QSIs provide hope for the development of such drugs.

Paeoniflorin combined with norfloxacin ameliorates drug-resistant Streptococcus suis infection.

BACKGROUND: The increased resistance of bacterial pathogens to fluoroquinolones (FQs), such as norfloxacin and ciprofloxacin, supports the need to develop new antibacterial drugs and combination therapies using conventional antibiotics. The LuxS/AI-2 quorum sensing (QS) system can regulate the complex group behaviour of Streptococcus suis and impact its susceptibility to FQs. OBJECTIVES: We investigated the combination of paeoniflorin and norfloxacin as a novel and effective strategy against FQ-resistant S. suis. METHODS: FIC, AI-2 activity assay, real-time RT-PCR and biofilm inhibition assays were performed to investigate the in vitro effect of paeoniflorin combined with norfloxacin. Mouse protection and mouse anti-infection assays were performed to investigate the in vivo effect of paeoniflorin combined with norfloxacin. RESULTS: FIC results showed that paeoniflorin and norfloxacin exert a synergistic bactericidal effect. Evidence was brought that paeoniflorin reduces the S. suis AI-2 activity and significantly down-regulates the transcription of the FQ efflux pump gene. In addition, paeoniflorin can inhibit biofilm formation, thereby promoting the ability of norfloxacin to kill S. suis. Finally, we showed in a mouse model that paeoniflorin in association with norfloxacin is effective to treat S. suis infections. CONCLUSIONS: This study highlighted the inhibitory potential of paeoniflorin on the LuxS/AI-2 QS system of S. suis, and provided evidence that it can inhibit the FQ efflux pump and prevent biofilm formation to cooperate with norfloxacin in the treatment of resistant S. suis-related infections.

Specific quantitative detection of Streptococcus suis and Actinobacillus pleuropneumoniae in co-infection and mixed biofilms.

Respiratory infections seriously affect the swine industry worldwide. Co-infections of two vital pathogenic bacteria Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae), colonizing the respiratory tract often occurs in veterinary clinical practice. Moreover, our previous research found that S. suis and A. pleuropneumoniae can form biofilm in vitro. The formation of a mixed biofilm not only causes persistent infections, but also increases the multiple drug resistance of bacteria, which brings difficulties to disease prevention and control. However, the methods for detecting S. suis and A. pleuropneumoniae in co-infection and biofilm are immature. Therefore, in this study, primers and probes were designed based on the conservative sequence of S. suis gdh gene and A. pleuropneumoniae apxIVA gene. Then, a TaqMan duplex real-time PCR method for simultaneous detection of S. suis and A. pleuropneumoniae was successfully established via optimizing the reaction system and conditions. The specificity analysis results showed that this TaqMan real-time PCR method had strong specificity and high reliability. The sensitivity test results showed that the minimum detection concentration of S. suis and A. pleuropneumoniae recombinant plasmid was 10 copies/µL, which is 100 times more sensitive than conventional PCR methods. The amplification efficiencies of S. suis and A. pleuropneumoniae were 95.9% and 104.4% with R2 value greater than 0.995, respectively. The slopes of the calibration curves of absolute cell abundance of S. suis and A. pleuropneumoniae were 1.02 and 1.09, respectively. The assays were applied to cultivated mixed biofilms and approximately 108 CFUs per biofilm were quantified when 108 CFUs planktonic bacteria of either S. suis or A. pleuropneumoniae were added to biofilms. In summary, this study developed a TaqMan real-time PCR assay for specific, accurate quantification of S. suis or A. pleuropneumoniae in mixed biofilms, which may help for the detection, prevention and control of diseases caused by a bacterial mixed infection involving S. suis and A. pleuropneumoniae.

Sub-Inhibitory Concentrations of Amoxicillin and Tylosin Affect the Biofilm Formation and Virulence of Streptococcus suis.

Streptococcus suis (S. suis) can form a protective biofilm during infection and lead to prolonged disease. Oral antibiotics are often used for treatment in clinical practice, but sub-inhibitory concentration levels often exist due to low oral absorption rate, resulting in disease deterioration. The purpose of this study was to investigate the effects of Amoxicillin and Tylosin on the biofilm formation and virulence of S. suis HA9801 at sub-inhibitory concentration. We first determined that the test groups (1/4MIC Amoxicillin and Tylosin) could significantly increase the amount of biofilm formation without affecting bacterial growth. The LD50 value of the test groups was significantly higher than that of the control group in the mouse infection model. In the mouse infection model, the LD50 value of the experimental group was significantly increased, but the tissue bacterial load was significantly decreased. Further RT-PCR analysis showed that the expression levels of virulence-related genes in the experimental group were significantly reduced. Our study suggests that both Amoxicillin and Tylosin at sub-inhibitory concentrations could enhance the biofilm formation ability of S. suis HA9801 and reduce its virulence to form persistent infection.

Contribution of quorum sensing to virulence and antibiotic resistance in zoonotic bacteria.

Quorum sensing (QS), which is a key part of cell/cell communication, is widely distributed in microorganisms, especially in bacteria. Bacteria can produce and detect the presence of QS signal molecule, perceive the composition and density of microorganisms in their complex habitat, and then dynamically regulate their own gene expression to adapt to their environment. Among the many traits controlled by QS in pathogenic bacteria is the expression of virulence factors and antibiotic resistance. Many pathogenic bacteria rely on QS to govern the production of virulence factors and express drug-resistance, especially in zoonotic bacteria. The threat of antibiotic resistant zoonotic bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Targeting QS has proven to be a promising alternative to conventional antibiotic for controlling infections. Here we review the QS systems in common zoonotic pathogenic bacteria and outline how QS may control the virulence and antibiotic resistance of zoonotic bacteria.

Structure and Signal Regulation Mechanism of Interspecies and Interkingdom Quorum Sensing System Receptors.

Quorum sensing (QS) is a signaling mechanism for cell-to-cell communication between bacteria, fungi, and even eukaryotic hosts such as plant and animal cells. Bacteria in real life do not exist as isolated organisms but are found in complex, dynamic, and microecological environments. The study of interspecies QS and interkingdom QS is a valuable approach for exploring bacteria-bacteria interactions and bacteria-host interaction mechanisms and has received considerable attention from researchers. The correct combination of QS signals and receptors is key to initiating the QS process. Compared with intraspecies QS, the signal regulation mechanism of interspecies QS and interkingdom QS is often more complicated, and the distribution of receptors is relatively wide. The present review focuses on the latest progress with respect to the distribution, structure, and signal transduction of interspecies and interkingdom QS receptors and provides a guide for the investigation of new QS receptors in the future.

Paeoniflorin reduce luxS/AI-2 system-controlled biofilm formation and virulence in Streptococcus suis.

Streptococcus suis (S. suis), more specifically serotype 2, is a bacterial pathogen that threatens the lives of pigs and humans. Like many other pathogens, S. suis exhibits quorum sensing (QS) system-controlled virulence factors, such as biofilm formation that complicates treatment. Therefore, impairing the QS involving LuxS/AI-2 cycle in S. suis, may be a promising alternative strategy for overcoming S. suis infections. In this study, we investigated paeoniflorin (PF), a monoterpenoid glycoside compound extracted from peony, as an inhibitor of S. suis LuxS/AI-2 system. At a sub-minimal inhibitory concentration (MIC) (1/16 MIC; 25 µg/ml), PF significantly reduced biofilm formation by S. suis through inhibition of extracellular polysaccharide (EPS) production, without affecting bacterial growth. Moreover, evidence was brought that PF reduces AI-2 activity in S. suis biofilm. Molecular docking indicated that LuxS may be the target of PF. Monitoring LuxS enzymatic activity confirmed that PF had a partial inhibitory effect. Finally, we showed that the use of PF in a mouse model can relieve S. suis infections. This study highlighted the anti-biofilm potential of PF against S. suis, and brought evidence that it may as an inhibitor of the LuxS/AI-2 system to prevent S. suis biofilm-related infections. PF can thus be used as a new type of natural biofilm inhibitor for clinical application.

Evaluation of immune effect of Streptococcus suis biofilm-associated protein PDH.

As a zoonotic pathogen, Streptococcus suis(S. suis) takes pigs as the main host and is mainly colonizes in the upper respiratory tract and tonsil of pigs, causing septicemia, endocarditis and meningitis in pigs. Pyruvate dehydrogenase (PDH) is an enzyme that catalyzes the conversion of pyruvate to acetyl-CoA. As an immunogenic membrane-associated protein in S. suis, it has been found to be closely related to the formation of biofilm. In this study, the recombinant PDH (rPDH) of S. suis ZY05719 (serotype 2) was expressed and purified in E. coli by His affinity chromatography. Western blotting analysis showed that there was a strong specific reaction between PDH protein and PDH antiserum. Mice were immunized with recombinant PDH and inactivated bacteria, and the relative survival rates were 70 % and 60 %, respectively. In addition, mice immunized with PDH caused high levels of antibodies and high expression of immune-related genes in the spleen, which significantly protected the liver, brain and spleen from pathological damage. In addition, PDH antiserum could significantly inhibit the growth of S. suis and the formation of S. suis biofilm in vitro. These results further suggest that PDH is a promising candidate for S. suis biofilm-related subunit vaccine.

mRNA-Seq reveals the quorum sensing system luxS gene contributes to the environmental fitness of Streptococcus suis type 2.

BACKGROUND: Streptococcus suis type 2 (SS2) is an important zoonotic pathogen. We have previously reported the structure of LuxS protein and found that the luxS gene is closely related to biofilm, virulence gene expression and drug resistance of SS2. However, the mechanism of luxS mediated SS2 stress response is unclear. Therefore, this experiment performed stress response to luxS mutant (ΔluxS) and complement strain (CΔluxS), overexpression strain (luxS+) and wild-type SS2 strain HA9801, and analyzed the differential phenotypes in combination with transcriptome data. RESULTS: The results indicate that the luxS gene deletion causes a wide range of phenotypic changes, including chain length. RNA sequencing identified 278 lx-regulated genes, of which 179 were up-regulated and 99 were down-regulated. Differential genes focus on bacterial growth, stress response, metabolic mechanisms and drug tolerance. Multiple mitotic genes were down-regulated; while the ABC transporter system genes, cobalamin /Fe3+-iron carrier ABC transporter ATPase and oxidative stress regulators were up-regulated. The inactivation of the luxS gene caused a significant reduction in the growth and survival in the acid (pH = 3.0, 4.0, 5.0) and iron (100 mM iron chelator 2,2'-dipyridyl) stress environments. However, the mutant strain ΔluxS showed increased antioxidant activity to H2O2 (58.8 mmol/L). CONCLUSIONS: The luxS gene in SS2 appears to play roles in iron metabolism and protective responses to acidic and oxidative environmental conditions.

The otc gene of Streptococcus suis plays an important role in biofilm formation, adhesion, and virulence in a murine model.

Streptococcus suis (S. suis) is an emerging zoonotic pathogen that can cause meningitis, arthritis, pneumonia, and sepsis. It poses a serious threat to the swine industry and public health worldwide. Ornithine carbamoyltransferase (OTC) is involved in the arginine deiminase system. OTC, which is a widely distributed enzyme in microorganisms, mammals, and higher plants, catalyzes the conversion of ornithine to citrulline. The present study showed that the otc gene plays an important role in the pathogenesis of S. suis infections. The ability of an otc-deficient mutant (Δotc) to form a biofilm was significantly reduced compared to the wild-type (WT) strain, as determined by crystal violet staining. Confocal laser scanning microscopy and scanning electron microscopy observations showed that the weakening of biofilm formation by the Δotc strain is related to a decrease in the extracellular matrix. In addition, compared to the WT strain, the Δotc strain had a reduced capacity to adhere to human laryngeal epidermoid carcinoma (HEp-2) cells compared to the WT strain. A real-time PCR analysis showed that the expression of adhesion-related genes by the Δotc strain was also lower than that of the WT strain. The virulence of the Δotc strain was significantly lower than that of the WT strain in a murine infection model. In addition, a histological analysis showed that the pathogenicity of the Δotc strain was lower than that of the WT strain, causing only slight inflammatory lesions in lung, liver, spleen, and kidney tissues. No significant differences were observed between the complemented mutant (CΔotc) and WT strains with respect to biofilm formation, adhesion, gene expression, and virulence. The present study provided evidence that the otc gene plays a pivotal role in the regulation of S. suis adhesion and biofilm formation. It also suggested that the otc gene is indirectly involved in the pathogenesis of S. suis serotype 2 infections.

Reverse-transcription recombinase-aided amplification assay for H7 subtype avian influenza virus.

H7 subtype avian influenza virus infection is an emerging zoonosis in some Asian countries and an important avian disease worldwide. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. In this study, we developed a reverse-transcription recombinase-aided amplification assay for the detection of H7 subtype avian influenza virus. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus, which are the other main respiratory viruses affecting birds. The analytical sensitivity was 102 RNA copies per reaction at a 95% probability level according to probit regression analysis, with 100% specificity. Compared with published reverse-transcription quantitative real-time polymerase chain reaction assays, the κ value of the reverse-transcription recombinase-aided amplification assay in 342 avian clinical samples was 0.988 (p < .001). The sensitivity for avian clinical sample detection was 100% (95%CI, 90.40%-100%), and the specificity was 99.96% (95%CI, 97.83%-99.98%). These results indicated that our reverse-transcription recombinase-aided amplification assay may be a valuable tool for detecting avian influenza H7 subtype virus.

Non-polio enteroviruses from acute flaccid paralysis surveillance in Shandong Province, China, 1988-2013.

Enteroviruses (EVs) are important human pathogens associated with various clinical syndromes. This study represents an overview of non-polio enteroviruses (NPEVs) isolated from acute flaccid paralysis (AFP) surveillance in Shandong Province, China from 1988 to 2013. Altogether 792 and 170 NPEV isolates were isolated from stool specimens of 9263 AFP cases and 1059 contacts, respectively. Complete VP1 sequencing and typing on all 962 isolates revealed 53 NPEV types in which echovirus (E) 6 (7.6%), E14 (7.6%), E11 (7.4%), coxsackievirus (CV) B3 (7.4%), E25 (5.6%), CVB5 (4.9%), E7 (4.5%) and EV-A71 (4.4%) were the eight most commonly reported serotypes. Distinct summer-fall seasonality was observed, with June-October accounting for 79.3% of isolation from AFP cases with known month of specimen collection. Increase of isolation of EV-A71 and CVA--the predominant pathogens for the hand, foot, and mouth disease--was observed in recent years. Sequence analysis on VP1 coding region of EV-A71 and E6 suggested Shandong strains had great genetic divergence with isolates from other countries. The results described in this study provide valuable information on the circulation and emergence of different EV types in the context of limited EV surveillance in China.

[Study on the genetic characterization and molecular epidemiology of echovirus 11 strains isolated in Shandong province, China].

OBJECTIVE: To analyze the evolution and genetic characterization of echovirus 11 (Echo11) from the acute flaccid paralysis (AFP) cases in Shandong province. METHODS: Isolation of Enterovirus was performed from stool samples of AFP cases from 1994 to 2009. All positive strains were sero-typed by neutralization test. Entire VP1 coding region from 27 strains typed as Echo11 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), and subsequently phylogenetic analyse on VP1 sequences from these strains and others published in GenBank were conducted. RESULTS: From 1994 to 2009, altogether 915 non-polio enterovirus (NPEV) strains were isolated with 79 (8.6%) isolates serotyped as Echo11. There were 876 nucleotides (nt) in the complete VP1 genes, encoding 292 amino acids (aa). The nt identities were 76.4% - 100.0% among those Echo11 Shandong strains with the aa identities as 91.4% - 100.0%. The nt and aa identities were 77.7% - 80.7% and 90.7% - 94.8% between Shandong strains and prototype strains, respectively. CONCLUSION: All Echo11 strains could be divided into four genotypes. Shandong strains that forming three (A1, A2 and C1) new sub-genotypes, with every sub-genotype had several brands. Sub-genotype A1 appeared to be the lately circulating one.

The complete genome analysis of two enterovirus 96 strains isolated in China in 2005 and 2009.

Enterovirus 96 (EV96) is a new member of species Human Enterovirus C (HEV-C). In this report, genomic characterization of two EV96 strains isolated from acute flaccid paralysis surveillance in Shandong province of China in 2005 and 2009 is described. The two strains, designated 05517 and 09228C1, had 82.7% genomic similarity with each other and 75.1-84.2% with other three strains available from GenBank in complete genome sequences. In VP1 coding region, they had 77.6-86.6% nucleotide similarity with other EV96 strains. Interestingly, deletions of 3 nucleotides in the VP3 coding region of strain 09228C1, and of 3 nucleotides in the 3A region of both Shandong strains were observed. Simplot and bootscanning analysis on HEV-C genome sequences were performed, and evidence of recombination in P3 region for Shandong EV96 strains was found. In conclusion, these strains had distant genetic relationship with each other and with other EV96 strains.

[Genotype distribution of enterovirus group B isolated in Shandong Province, China].

In order to explore the genotype distribution of human enterovirus group B (HEV-B) in Shandong Province and to study the correlation between HEV-B serotypes and disease outbreaks, we sequenced and analyzed the entire VP1 coding region of HEV-B isolated from acute flaccid parolysis (AFP) system and disease outbreaks in Shandong province during 1998-2008. All together twenty nine HEV-B serotypes were identified, including twenty Echovirus (ECHO) serotypes, five Coxsackievirus B (CVB1-5) serotypes, one Coxsackievirus A9(CVA9) serotype, and newer enteroviruses EV73, EV75, and EV97. E11, CVB3, E6, E14 and E25 were the five frequently isolated serotypes from AFP surveillance system. CVB3, CVB5 and ECHO30 were the major causative agent of aseptic meningitis in Shandong province. Comparison of nucleotide sequence homology showed 75.4%-99.6% inter-typic identities within Shandong strains, and 73.8%-85.2% identities with prototype strains. Amino acid sequence comparison showed the differentiation was not much. Our research showed different serotypes possessed distinct time-cycling pattern, and different sub-genotypes could be further classified according to the inter-typic genetic distance. Thereby the route and range of transmission of HEV could be determined.