Expression and clinical significance of pattern recognition receptor-associated genes in hand, foot and mouth disease

Objective:To explore which pattern recognition receptors(PRRs)play a key role in the development of hand,foot,and mouth disease(HFMD)by analyzing PRR-associated genes.Methods:We conducted a comparative analysis of PRR-associated gene expression in human peripheral blood mononuclear cells(PBMCs)infected with enterovirus 71(EV-A71)which were derived from patients with HFMD of different severities and at different stages.A total of 30 PRR-associated genes were identified as significantly upregulated both over time and across different EV-A71 isolates.Subsequently,ELISA was employed to quantify the expression of the six most prominent genes among these 30 identified genes,specifically,BST2,IRF7,IFI16,TRIM21,MX1,and DDX58.Results:Compared with those at the recovery stage,the expression levels of BST2(P=0.027),IFI16(P=0.016),MX1(P=0.046)and DDX58(P=0.008)in the acute stage of infection were significantly upregulated,while no significant difference in the expression levels of IRF7(P=0.495)and TRIM21(P=0.071)was found between different stages of the disease.The expression levels of BST2,IRF7,IFI16 and MX1 were significantly higher in children infected with single pathogen than those infected with mixed pathogens,and BST2,IRF7,IFI16 and MX1 expression levels were significantly lower in coxsackie B virus(COXB)positive patients than the negative patients.Expression levels of one or more of BST2,IRF7,IFI16,TRIM21,MX1 and DDX58 genes were correlated with PCT levels,various white blood cell counts,and serum antibody levels that reflect disease course of HFMD.Aspartate aminotransferase was correlated with BST2,MX1 and DDX58 expression levels.Conclusions:PRR-associated genes likely initiate the immune response in patients at the acute stage of HFMD.

Transmission Dynamics and Optimal Control Strategies of a Hand-Foot-Mouth Disease Model with Treatment and Vaccination Interventions

In this article, the transmission dynamics of a Hand-Foot-Mouth disease model with treatment and vaccination interventions are studied. We calculated the basic reproduction number and proved the global stability of disease-free equilibrium when R0 R0 > 1. Meanwhile, we obtained the optimal control strategies minimizing the cost of intervention and minimizing the infected person. We also give some numerical simulations to verify our theoretical results.

Computational Investigation of Hand Foot Mouth Disease Dynamics with Fuzziness

The first major outbreak of the severely complicated hand,foot and mouth disease(HFMD),primarily caused by enterovirus 71,was reported in Taiwan in 1998.HFMD surveillance is needed to assess the spread of HFMD.The parameters we use in mathematical models are usually classical mathematical parameters,called crisp parameters,which are taken for granted.But any biological or physical phenomenon is best explained by uncertainty.To represent a realistic situation in any mathematical model,fuzzy parameters can be very useful.Many articles have been published on how to control and prevent HFMD from the perspective of public health and statistical modeling.However,few works use fuzzy theory in building models to simulateHFMDdynamics.In this context,we examined anHFMD model with fuzzy parameters.A Non Standard Finite Difference(NSFD)scheme is developed to solve the model.The developed technique retains essential properties such as positivity and dynamic consistency.Numerical simulations are presented to support the analytical results.The convergence and consistency of the proposed method are also discussed.The proposed method converges unconditionally while the many classical methods in the literature do not possess this property.In this regard,our proposed method can be considered as a reliable tool for studying the dynamics of HFMD.

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

Construction of Plant Expression Vector for Hand,Foot and Mouth Virus EV71-VP1 Gene and Its Expression in Tomato

EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.

Surveillance of Hand,Foot,and Mouth Disease in China's Mainland (2008-2009)

Objective Since HFMD was designated as a class C communicable disease in May 2008,18 months surveillance data have been accumulated to December 2009.This article was to describe the distribution of HFMD for age,sex,area,and time between 2008 and 2009,to reveal the characteristics of the epidemic.Methods We analyzed weekly reported cases of HFMD from May 2008 to December 2009,and presented data on the distribution of age,sex,area and time.A discrete Poisson model was used to detect spatial-temporal clusters of HFMD.Results More than 1 065 000 cases of HFMD were reported in China's Mainland from May 2008 to December 2009 （total incidence：12.47 per 10 000）.Male incidence was higher than female for all ages and 91.9% of patients were 5 years old.The incidence was highest in Beijing,Shanghai,Zhejiang and Hainan.The highest peak of HFMD cases was in April and the number of cases remained high from April to August.The spatial-temporal distribution detected four clusters.Conclusion Children 5 years old were susceptible to HFMD and we should be aware of their vulnerability.The incidence was higher in urban than rural areas,and an annual pandemic usually starts in April.

Characterization of Hand,Foot,and Mouth Disease in China between 2008 and 2009

Objective To investigate the epidemiological and cl （HFMD） since several outbreaks of HFMD caused between 2007 and 2008. inical features of hand, foot and mouth disease by enteroviruses were documented in China Methods HFMD cases reported to the National Infectious Disease Information Management System database between May 2008 and April 2009 were assessed. Clinical features in some of the severe and fatal cases were analyzed the etiology of the outbreaks was investigated. Results 89.1% of reported HFMD cases were found in children〈5 year-old with an age-specific incidence rate of 834.1/100 000 in the first year as the notifiable disease in China from May 2008 to April 2009. The incidence, mortality and percentage of severe cases were studied for three regions of China and found to be highest in the central region. The incidence of severe cases and mortality in rural population were significantly higher than those in urban population. Among the laboratory confirmed EV17 positive cases there were 52.6% mild, 83.5% severe, and 96.1% fatal cases. More myoclonic jerks were found in the severe case group than in group that died. Tachypnea, lip purpling, pink foaming and low limb temperature occurred more frequently in the fatal cases than in the severe cases. Conclusion The epidemic of HFMD in China was characterized predominantly by EV71 infections, had relatively high mortality rates especially in the central region, and was most prevalent in young, rural populations.

Using Interrupted Time Series Design to Analyze Changes in Hand, Foot, and Mouth Disease Incidence during the Declining Incidence Periods of 2008-2010 in China

Objective To identify patterns of hand, foot and mouth disease （HFMD） incidence in China during declining incidence periods of 2008, 2009, and 2010. Methods Reported HFMD cases over a period of 25 months were extracted from the National Disease Reporting System （NDRS） and analyzed. An interrupted time series （ITS） technique was used to detect changes in HFMD incidence rates in terms of level and slope between declining incidence periods of the three years. Results Over 3.58 million HFMD cases younger than 5 years were reported to the NDRS between May 1, 2008, and May 31, 2011. Males comprised 63.4% of the cases. ITS analyses demonstrated a significant increase in incidence rate level （P〈0.0001） when comparing the current period with the previous period. There were significant changes in declining slopes when comparing 2010 to 2009, and 2010 to 2008 （all P〈O.O05）, but not 2009 to 2008. Conclusion Incremental changes in incidence rate level during the declining incidence periods of 2009 and 2010 can potentially be attributed to a few factors. The more steeply declining slope in 2010 compared with previous years could be ascribed to the implementation of more effective interventions and preventive strategies in 2010. Further investigation is required to examine this possibility.

Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like acute flaccid paralysis. A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997, and caused 41 deaths amongst young children. In late 2000, a recurrence of an outbreak of HFMD occurred in Malaysia with 8 fatalities in peninsular Malaysia. Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities. A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006. The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010. As with other HFMD outbreaks in Malaysia, both EV71 and CA16 were the main aetiological viruses isolated. In similarity with the HFMD outbreak in 2005, the isolation of CA16 preceded the appearance of EV71. Based on the VP1 gene nucleotide sequences, 4 sub-genogroups of EV71 (C1, C2, B3 and B4) co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak. EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003. In the 2005 outbreak, besides the EV71 strains of sub-genogroup C1, EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5. Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia. Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years. In each of the past outbreaks, more than one sub-genogroup of the virus co-circulate.

Molecular Epidemiological Analysis of Echovirus 19 Isolated From an Outbreak Associated With Hand, Foot, and Mouth Disease (HFMD) in Shandong Province of China

To elucidate the genetic characterization and molecular epidemiological features of Echovirus 19 （El9） isolates collected from an outbreak associated with hand, foot and mouth disease （HFMD） in Tai＇an city of Shandong Province of China from July to September, 2003. Methods Thirty seven Echovirus 19 isolates were isolated from stool specimens and throat swabs collected during the outbreak, then major capsid （VP1） genomic sequence was determined, and phylogenetic tree was done based on the VP1 sequences among these 37 and other El9 viruses deposited in the Genbank. Also a representative strain named CHN-SD03-TN12 was selected for sequencing of 5′-untranslated regions （5′-UTR）. Results The identity rate was about 98.9%-100% among all these 37 El9 viruses. The genetic relationships between these 37 El9 isolates and other strains reported were also depicted. The identity rate was about 78.4%-78.9% compared with El9 reference strain Burke. The substitutions in the sequence of 5′-UTR resulted in changes in the conjectural properties of 5′-UTR of El9 viruses. Condusion The genetic features of El9 viruses isolated during the outbreak in Shandong Province in 2003 may be associated with a genetic and antigenic drift that changes the virulence of the Shandong isolates, but the molecular changes in Shandong El9 viruses contributing to their phenotype remain to be further illuminated. However, the sequences described in this paper substantiate the changes taken place in capsid VPI and 5′UTR regions. These substitutions may contribute to their tropism and virulence, and play a significant role in pathogenesis and clinical manifestations of the disease.

Molecular Epidemiology of Enteroviruses Associated with Hand, Foot, and Mouth Disease in the Mainland of China

Hand, foot, and mouth disease （HFMD） is a common contagious illness which occurs worldwide both sporadically and in epidemics. The disease mainly affects, children and the typical symptoms, which may resolve spontaneously, include mucocutaneous papulovesicular lesions on the hands, feet, mouth, and buttocks. In rare cases, however, the patients may also develop neurological complications such as neurogenic pulmonary edema,

Coxsackievirus A6 was the most common enterovirus serotype causing hand,foot,and mouth disease in Shiyan City,central China

BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following the introduction of EV-A71 vaccines in China since 2016,the situation could change.CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China.However,the epidemiological characteristics of central China remain unknown.AIM To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City,central China,in recent years.METHODS The epidemiological,clinical,and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed.196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020.To detect and genotype enteroviruses,real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used.In Shiyan,168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes.Based on the logistic regression model,the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan.RESULTS From 2016 to 2020,35840 HFMD cases were reported in Shiyan.The number of cases decreased by 48.4%from 2016 to 2017.Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year,respectively.In 2020,a decrease of about 85.5%was reported when compared to 2019.The most common serotypes shifted from EV-A71 and CV-A16(about 60%-80%in 2016 and 2018)to others(more than 80.0%in 2017,2019,and 2020).EV-A71 lost its dominance in 2017 in Shiyan.Among 196 confirmed HFMD cases,85.7%tested positive for enterovirus,with CV-A6 being the most common serotype(121/168,72.0%).The positive rates for CV-A16 and CVA10 were 4.8%and 3.0%,respectively.There was no EV-A71 discovered.Infection with CV-A6 was linked to fever,myocardial damage,increased creatine kinase MB isoenzyme,and lactate dehydrogenase levels.CONCLUSION CV-A6 was the most common enterovirus serotype in Shiyan City,replacing EV-A71 and CV-A16 as the HFMD pathogen.Developing vaccines against CV-A6 or multiple pathogens,as well as rising CV-A6 surveillance,will help prevent HFMD in central China.

Clinical and neuroimaging features of enterovirus71 related acute flaccid paralysis in patients with hand-foot-mouth disease

Objective:To investigate clinical and neuroimaging features of enterovirus71(EV71) related acute flaccid paralysis in patients with hand-fool-mouth disease.Methods:Nine patients with acute flaccid paralysis met the criterion of EV71 induced hand-foot-mouth disease underwent spinal and brain MR imaging from May 2008 to Sep 2012.Results:One extremity flaccid was found in four cases(3 with lower limb,1 with upper limb),two limbs flaccid in three cases(2 with lower limbs,1 with upper limbs),and four limbs flaccid in two cases.Spinal MRI studies showed lesion with high signal in T2-weighted images(T2WI) and low signal T1-weighted images(T1WI) in the spinal cord of all nine cases,and the lesions were mainly in bilateral and unilateral anterior hom of cervical spinal cord and spinal cord below thoracic 9(T9) level.In addition,the midbrain,pons, and medulla,which were involved in 3 cases with brainstem encephalitis,demonstrated abnormal signal.Moreover,spinal cord contrast MRI studies showed mild enhancement in corresponding anterior hom of the involved side,and strong enhancement in its ventral root.Conclusions: EV71 related acute flaccid paralysis in patients with hand-foot-mouth disease mainly affected the anterior hom regions and ventral root of cervical spinal cord and spinal cord below T9 level. MR imaging could efficiendy show the characteristic pattern and extent of the lesions which correlated well with the clinical features.

Spatial Trend of Foot and Mouth Disease Virus (FMDV) Serotypes in Cattle and Buffaloes, Pakistan

The present study describes the frequency of Foot and Mouth Disease （FMD） virus serotypes （O, A and Asia-l） in major regions （all provinces） of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples （Epithelial tissue） have been analyzed during a period of five years （2005-2009）. Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ＇O＇ serotype caused most outbreaks （20.7 %）, followed by serotype A （6.6 %） and serotype Asia-1 （4.6 %） while there was no positive case of type ＇C＇. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples （〉50 outbreaks）, the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1%, higher than in buffalo （28.7 %）. There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I （2005-2007）, there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II （2008-2009）, the overall prevalence was 59.21%, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.

Comparison of transient and stable expression of foot-and-mouth disease virus capsid proteins in mammalian cells

Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

Establishment of Indirect ELISA Diagnosis Technique based on the VP1 Protein of Foot and Mouth Disease Virus Serotype A

The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.

Impact of Vaccination and Culling on Controlling Foot and Mouth Disease: A Mathematical Modelling Approach

Foot and mouth disease (FMD) is a major threat, not only to countries whose economies rely on agricultural exports, but also to industrialized countries that maintain a healthy domestic livestock industry by eliminating major infectious diseases from their livestock populations. In this paper a simple mathematical model is formulated and comprehensively analyzed to assess the impact of vaccination and culling on controlling FMD. Overally the study demonstrates that vaccination and culling are essential on controlling FMD if they are all implemented. Furthermore the study illustrates that culling latently infected (early detection of infected animals) is extremely important on controlling FMD dynamics.

Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.

The Complete Genomic Sequence of a Foot-and-Mouth Disease Virus Isolated from the Swine

The complete genomic sequence of foot-and-mouth disease virus (FMDV) Chinese strain OH/CHA/99 was determined. The 8040 nt sequence and the deduced amino acid sequence werecompared with FMDV sequences published. The results showed that OH/CHA/99 shared highersequence homology with OTYTW/97, indicating their close genetic relationship. However,the strain had lower sequence identity with O1/Kaufbeuren/66 strain. Besides, largedeletions in 3A coding region were observed in OH/CHA/99. It was shown that the poly (A)tail of OH/CHA/99 had 56 As at least.