Experimental infection of foot and mouth disease virus (FMDV) upregulates the expression of Coxsackie and adenovirus receptor (CAR) in the myocardium of suckling mice.

The relative overexpression of Coxsackie and adenoviral receptor (CAR) predisposes children to viral myocarditis. As the foot and mouth disease virus (FMDV) causes fatal myocarditis in calves, lambs, and piglets and belongs to the same family as the Coxsackie virus, we investigated the role of CAR in FMDV induced myocarditis in the suckling mice model. Swiss albino suckling mice of 5 days (n = 24) were divided into two equal groups. One group was inoculated with suckling mice adapted FMDV serotype O at 10 LD50, while the other group served as uninfected control. In addition, adult mice (n = 12) served as the control for age related CAR expression and lack of pathogenicity to FMDV. The establishment of myocarditis was confirmed by histopathological changes typical of myocarditis along with immunolocalization of FMDV antigens in the heart of suckling mice. The FMDV inoculated suckling mice group showed a significant upregulation of CAR transcripts by 2.5 folds, overexpression of CAR protein by densitometric analysis of immunoblots, and intense immunolocalization of CAR in the sarcolemma and intercalated discs of cardiomyocytes as compared to the uninfected suckling mice group and adult mice. It was concluded that FMDV infection induced overexpression of CAR in the myocardium of suckling mice.

Production of polyclonal viperin antisera using N-terminal deleted recombinant bovine viperin.

Viperin, also known as radical S-adenosyl methionine domain-containing protein (RSAD2) is a multifunctional interferon-stimulated gene (ISG) that is activated during the viral infections. Viperin belongs to S-adenosyl methionine (SAM) superfamily of enzymes known to catalyze radical-mediated reactions and viperin inhibits a wide range of DNA and RNA viruses through its broad range of activity. The present study reports cloning and expression of bovine viperin in a bacterial expression system. PCR-based site-directed mutagenesis was carried out for deletion of N-terminal 1-70 amino acid containing amphipathic helix of viperin that interferes in protein expression and purification. The resultant truncated viperin protein was expressed in Escherichia coli, BL-21(DE3) competent cells and purified using nickel charged affinity column. The truncated 54 kDa protein was confirmed by western blot using human RSAD2 as a probe. Further, in house, hyperimmune serum was raised against the truncated viperin in the rabbit and the reactivity was confirmed by western blot using mammalian expression vector construct of viperin transfected in Baby Hamster kidney (BHK) cells and in MDBK cells infected with Foot and Mouth disease Asia I virus.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Characterization of arrhythmias, evaluation of cardiac biomarkers and their association with survival in calves suffering from foot-and-mouth disease.

INTRODUCTION: Foot-and-mouth disease (FMD) causes mortality in calves due to myocarditis; however, the effects of FMD virus on cardiac arrhythmogenesis and Purkinje cells are unknown. Identifying diagnostic and prognostic markers in FMD-affected calves may be useful in disease management in the endemic countries. MATERIALS AND METHODS: A total of 81 FMD-affected calves were prospectively monitored till death or recovery. Foot-and-mouth disease was diagnosed by serology and reverse transcriptase-polymerase chain reaction (RT-PCR). Electrocardiography was recorded and serum cardiac biomarkers were measured. Histopathological examination of the ventricular myocardium was carried out in the calves that died of FMD (n = 33). Apparently healthy calves (n = 15) served as control. RESULTS: Serology and RT-PCR consistently revealed that the FMD was caused by serotype O virus. Arrhythmias occurred in 62 of 81 (76.5%) FMD-affected calves, of which, ventricular premature complexes (VPCs) were the most common type (22%). The combined mortality rate due to ventricular tachycardia, polymorphic VPCs, and atrial fibrillation was 27.6%. Receiver operating characteristic curve analysis revealed that cardiac troponin I (cTnI) concentrations of ≥1.3 ng/mL were diagnostic of myocarditis with a sensitivity and specificity of 90% and 100%, respectively. Similarly, serum cTnI concentrations of <6.4 ng/mL were a good predictor of survival [odds ratio of 263; 95% confidence interval: 29-2371]. Histopathology of the myocardium revealed hyaline degeneration, necrosis, edema, mononuclear cell infiltration, and disruption by fibroblasts. Atrophy of the Purkinje cells was also present. CONCLUSIONS: FMD induces cardiac arrhythmias and Purkinje cell pathology in the calf. Portable ECG coupled with assay of serum cTnI would help in predicting survival in FMD-affected calves.

Hematological and serum biochemical profile in cattle experimentally infected with foot-and-mouth disease virus.

BACKGROUND AND AIM: Foot-and-mouth disease (FMD) is an acute viral infection affecting cloven-hoofed animals causing vesicular erosions in the oral cavity and interdigital space. The present study was undertaken to ascertain the time-dependent changes in clinical, hematological, and biochemical profiles in different breeds of cattle following experimental infection. MATERIALS AND METHODS: The animals were inoculated with 1.0×104 50% bovine tongue infectious dose (BTID50) by intradermolingual route. Clinical signs were observed, and blood/serum samples were collected at different time intervals. RESULTS: The white blood cell count declined sharply on days 7-13 and recovered on day 14 post-FMD infection. Biochemical analysis of serum markers for vital organ profile revealed no marked damage. However, a significant increase in blood urea nitrogen (BUN) value indicated pre-renal azotemia. Transient hyperthyroidism was indicated by the rise in T3 and T4 that can be correlated with a decrease in triglyceride and total cholesterol levels. In the cardiac damage assessment study, a distinct breed difference was observed wherein Malnad Gidda calves showed no cardiac damage. CONCLUSION: Except thyroid profile, BUN, and creatine kinase-myocardial band, all other serum biochemical parameters showed no significant abnormalities, whereas lymphopenia is the only hematological change and it is suggested that effective ameliorative measures should be targeted mainly on the feed/water intake, thyroid gland, and the level of lymphocytes.

Foot and mouth disease virus undergoes non-progressive replication in mice peritoneal macrophages and induces M1 polarization.

Despite the fact that macrophages link the innate and adaptive arms of immunity, it's role in the early infection of foot and mouth disease virus (FMDV) is largely unknown. Recently, depletion of macrophages in vivo after vaccination has shown to drastically diminish the protection against FMDV challenge in mouse model. Even the ability of macrophages to reduce or resist FMDV infection is not known hitherto. Therefore, we examined the replication ability of FMDV in mice peritoneal macrophages and the responsiveness in terms of macrophage polarization and cytokine production. Negative strand specific RT-PCR indicated replication of FMDV RNA in macrophages. Absolute quantitation of FMDV transcripts, immunofluorescence studies and titre of the infectious progeny virus revealed that replication peaked at 12 hpi and significantly declined by 18 hpi indicating non-progressive replication in the infected macrophages. Further, significant up regulation of inducible nitric oxide synthase by 8 -12 hpi and increase of M1 specific CD11c + cells by 42.6 % after infection showed that FMDV induce M1 polarization. A significant up regulation of TNFα and IL12 transcripts at 8 hpi supported that M1 macrophages were functional. Further, we studied the expression of Type I to III interferons (IFN) and other antiviral molecules. The results indicate a marked up regulation of Type I IFNα and ß by 9.2 and 11.2 fold, respectively at 8 hpi. Of the four IFN stimulated genes (ISG), viperin showed a significant up regulation by 286-fold at 12 hpi in the mice macrophages. In conclusion, the results suggest that replication of FMDV in mice peritoneal macrophages is non-progressive with up regulation of Type I IFN and ISGs. Further, FMDV induces M1 polarization in murine peritoneal macrophages.

Comparison of chemical extraction methods for determination of 146S content in foot-and-mouth disease oil-adjuvanted vaccine.

AIMS: To compare antigen extraction efficiency of chemical methods such as benzyl alcohol, chloroform, sodium citrate, extraction buffer with Tween-20 (EBT) and isopropyl myristate for determination of 146S content in the fresh and stored FMD oil-adjuvanted vaccines. METHODS AND RESULTS: Standard vaccine with antigen payload of 10, 5 and 5 µg per cattle dose (2 ml) for serotypes O, A and Asia1, respectively, was used to compare the antigen extraction efficiency of five chemical methods: benzyl alcohol, chloroform, sodium citrate, EBT buffer and isopropyl myristate. The purity of the extracted 146S antigen was quantified by caesium chloride (CsCl) ultracentrifugation. Serotype-specific sandwich ELISA (sELISA) was developed to identify the serotype and to compare the 146S in aqueous phase and ultrafractions. The antigen recovery was also tested in stored trivalent vaccine. Coefficient of regression was calculated to assess the predictive power of the benzyl alcohol extraction method. Of the five methods, benzyl alcohol showed consistent antigen recovery of >90% in monovalent as well as trivalent vaccines. Ultrafraction showed a 1·4 ratio at A259/239 nm in UV spectrophotometry indicating the presence of 146S. sELISA revealed that the antigen recovery was significantly less in ultrafractions than that of aqueous phase. Further, there was no significant difference in antigen recovery from stored trivalent vaccine for 12 months, indicating the usefulness of the benzyl alcohol method. Linear regression model revealed R2  = 0·99 with a narrow band of predictive interval. CONCLUSIONS: The benzyl alcohol method was efficient in extracting 146S from the monovalent and trivalent fresh and stored FMD vaccines. CsCl density gradient precisely quantified the 146S, while sELISA identified the serotype of the vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: When the benzyl alcohol method is coupled with CsCl density gradient and sELISA, it has the potential to determine the 146S content of FMD vaccine.

Generation of acid resistant virus like particles of vaccine strains of foot-and-mouth disease virus (FMDV).

Foot-and-mouth disease (FMD) is a contagious viral disease affecting cloven hoofed livestock. Insect cell expressed virus like particles (VLPs) are potential alternative to overcome the limitations of inactivated vaccine. However, at pH < 6.5, virus particles disassociate into pentameric structure resulting in loss of antigenicity. Accordingly, we generated seven mutant VLPs containing mutations in the structural genes of FMDV vaccine strains (N17D and/or H145Y for serotypes O/IND/R2/75 and Asia1/IND/63/72; and H142D for serotype A/IND/40/00) by PCR based site directed mutagenesis. Acid resistant VLPs produced by baculovirus expression system were tested for acid stability at pH 7.5, 6.5, 6.0 and 5.5 followed by reactivity in sandwich-ELISA (s-ELISA), which revealed mutant-1 (N17D) of serotype O and Asia1 retained the antigenicity in s-ELISA even at pH 5.5 as compared to other VLPs and wild-types. Further, the 75S empty capsids obtained in sucrose density gradient, when tested in liquid phase blocking ELISA (LPBE) in comparison to cell culture antigen indicated that the VLPs were stable at acidic pH. Transmission electron microscopy of OM-1 confirmed the intact morphology of the empty VLPs. It is concluded that acid resistant VLPs could be useful for developing new generation vaccine or diagnostic for FMDV.

Successful treatment of cardiac dysrhythmia associated with foot and mouth disease in a calf.

BACKGROUND: Foot and mouth disease (FMD), which causes myocarditis, results in 50% sudden death in the suckling calves. Occurrence of arrhythmias associated with FMD induced myocarditis in calves is not reported hitherto. The present work documents the arrhythmias associated with FMD in calf and their treatment using appropriate antiarrhythmic drugs. CASE DESCRIPTION: A three -month-old male Holstein Friesian crossbred calf naturally suffering from FMD was selected for the present study. FINDINGS/TREATMENT AND OUTCOME: Cardiac auscultation revealed grade 4 systolic murmurs and electrocardiography (ECG) showed sustained polymorphic ventricular premature complexes (PVPCs) with tachycardia on bipolar base apex lead. Apart from standard treatment, lidocaine 2% was administered at dose of 0.6 mg/kg intravenously over 15 min once a day and sinus rhythm was restored by 76 h post-treatment. Review of ECG and haematobiochemical examination revealed normal findings on 7th day of treatment. CONCLUSION: The study demonstrates the presence of sustained PVPCs with tachycardia due to FMD induced myocarditis and the successful use of lidocaine in restoring the sinus rhythm and recovery of the calf.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

Expression and purification of biologically active bovine Interferon λ3 (IL28B) in Pichia pastoris.

Interferon lambda-3 (IFNλ3) which is also known as IL28B is a member of type III Interferons which are structurally and genetically different from type I Interferons. These Interferons induce signal transduction pathways similar to type I Interferons which results in the activation of Interferon Stimulated Genes (ISGs). This group of Interferons are tissue specific and reported to have antiviral activity. In the present communication, we report the expression of bovine IFNλ3 gene (coding for the mature protein) in Pichia pastoris, purification of the expressed protein and evaluation of its biological activity. About 19 kDa protein expressed by the transformed Pichia cells, secreted into the media and the protein was purified by SP-Sepharose ion exchange chromatography with NaCl stepwise gradient elution. Specificity of the protein was confirmed by Western blotting. Pichia expressed IFNλ3 was found to be biologically active, as it induced ISGs (Mx protein, OAS and PKR genes) in bovine PBMCs. Further it was also found to modulate Th1/Th2 cytokines expression in the stimulated bovine PBMCs.

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

A competitive dipstick enzyme immunoassay for diagnosis of early pregnancy in bovine.

Early pregnancy diagnosis in bovines is one of the important aspects in efficient dairy farm management. In order to develop a competitive enzyme immunoassay (EIA) employing low cost reagents, anti-progesterone antiserum and progesterone-penicillinase enzyme conjugate were prepared. Using this anti-serum and conjugate along with pencillinV-starch-iodine substrate system, the competitive EIA was standardized. In the experiment, danazol, a weak androgen used to extract the progesterone bound to proteins in milk, was included after standardizing the optimum concentration. Incubation period and temperature and pH of the reaction mixture were also optimized. The developed test was validated with milk samples obtained from dairy farm and individual animal owners. Confirmation of the pregnancy was made by per rectum examination of the genital tract around 60 days post-insemination. The user friendly test procedure showed sensitivity and specificity of 83.3% and 87.5%, respectively as compared with residual binding method which was earlier developed in the laboratory with sensitivity and specificity of 100% and 87.5% respectively.