Comparison of the sensitivity of three cell cultures to ORFV.

BACKGROUND: Contagious ecthyma (CE) appears in the countries and regions containing goat and sheep farms, and it is considered a global epidemic. CE not only severely endangers the healthy development of the sheep and goat industries but also threatens human health. For viral infectious diseases, fast and effective isolation and culture of the pathogen is critical for CE diagnosis, and for disease prevention and control. Therefore, the sensitivity of bovine Sertoli cells to ORFV was estimate in this study. RESULTS: The sensitivities of bovine Sertoli cells, primary neonatal bovine testicular cells, and Madin-Darby bovine kidney (MDBK) cell line to ORFV were compared. Our results showed that the isolated bovine Sertoli cells were sensitive to inoculated ORFV, and viral titers were approximately 1 log higher than those in primary neonatal bovine testicular cells and in MDBK cell lines. CONCLUSION: Appropriately sensitive cells for the highly efficient isolation and culture of the ORFV were obtained. Culture of ORFV using the Sertoli cells showed good consistency and stability and also avoided the risk of other pathogens presenting during viral culture using a primary cell line. In addition, using these passaged bovine Sertoli cells to proliferate ORFV may simplify the CE diagnosis process, thereby reducing detection time and cost. Hence, this test has important practical significance for the diagnosis of CE and the research on the pathogenic mechanism of ORFV.

Development of an isothermoal amplification-based assay for rapid visual detection of an Orf virus.

BACKGROUND: Orf virus (ORFV) is the causative agent of a severe infectious skin disease (also known as contagious ecthyma) in goats, sheep and other small ruminants. Importantly, ORFV also infect humans which causes a public health concern in the context of changing environment and increase in human populations. The rapid detection is critical in effective control of the disease and urgently needed. RESULTS: A novel "point of care" molecular amplification assay for rapid visual detection of ORFV was developed based on isothermoal recombinase polymerase amplification (RPA) technology in combination with a simpler lateral flow immunoassay strip (ORFV RPA- LFD assay). The developed ORFV RPA- LFD assay was able to detect ORFV in less than 25 min. This assay was highly sensitive, with detection limit of as low as 80 copies/reaction, and highly specific, with no cross-reactions with capripox virus, foot-and-mouth disease virus and peste des petits ruminants virus. Furthermore, the ORFV RPA- LFD assay has good correlation with qPCR assay for detection of ORFV present in clinical samples. CONCLUSIONS: The developed ORFV RPA-LFD assay was a sensitive and specific method for rapid detection of ORFV, and has great potential as an onsite molecular diagnostic tool in control of Orf.

Review on the identification and role of Toxoplasma gondii antigenic epitopes.

Toxoplasma gondii is an obligate intracellular protozoan parasite with a broad range of hosts, and it causes severe toxoplasmasis in both humans and animals. It is well known that the progression and severity of a disease depend on the immunological status of the host. Immunological studies on antigens indicate that antigens do not exert their functions through the entire protein molecule, but instead, specific epitopes are responsible for the immune response. Protein antigens not only contain epitope structures used by B, T, cytotoxic T lymphocyte (CTL), and NK cells to mediate immunological responses but can also contain structures that are unfavorable for protective immunity. Therefore, the study of antigenic epitopes from T. gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology but it also plays a significant role in the development of new diagnostic reagents and vaccines. In this review, we summarized the immune mechanisms induced by antigen epitopes and the latest advances in identifying T. gondii antigen epitopes. Particular attention was paid to the potential clinical usefulness of epitopes in this context. Through a critical analysis of the current state of knowledge, we elucidated the latest data concerning the biological effects of epitopes and the immune results aimed at the development of future epitope-based applications, such as vaccines and diagnostic reagents.

Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6.

The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.

How foot-and-mouth disease virus receptor mediates foot-and-mouth disease virus infection.

This study reviews the FMDV receptor-binding domain, integrin receptors, and heparan sulfate receptors to provide references for studies regarding the mechanisms underlying FMDV infection.

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

BACKGROUND: Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. RESULTS: In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. CONCLUSIONS: These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Ssc-miR-7139-3p suppresses foot-and-mouth disease virus replication by promoting degradation of 3Cpro through targeting apoptosis-negative regulatory gene Bcl-2.

Foot-and-mouth disease is a highly contagious and infectious disease affecting cloven-hoofed animals. Disease control is complicated by its highly contagious nature and antigenic diversity. Host microRNAs (miRNAs) are post-transcriptional regulators that either promote or repress viral replications in virus infection. In the present study, we found that ssc-miR-7139-3p (Sus scrofa miR-7139-3p) was significantly up-regulated in host cells during foot-and-mouth disease virus (FMDV) infection. Overexpression of miR-7139-3p attenuated FMDV replication, whereas inhibition promoted FMDV replication. In addition, the survival rate of FMDV infected suckling mice was increased through injection of miR-7139-3p agomiR. Further studies revealed that miR-7139-3p targets Bcl-2 to initiate the apoptotic pathway and caspase-3 cleaved 3Cpro behind the 174th aspartic acid (D174), which eventually promotes the degradation of 3Cpro. Overall, our findings demonstrate that miR-7139-3p suppresses FMDV replication by promoting degradation of 3Cpro through targeting the apoptosis-negative regulatory gene Bcl-2.

Comparison of a loop-mediated isothermal amplification for orf virus with quantitative real-time PCR.

BACKGROUND: Orf virus (ORFV) causes orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. Therefore, a rapid, highly specific and accurate method for the diagnosis of ORFV infections is essential to ensure that the appropriate treatments are administered and to reduce economic losses. METHODS: A loop-mediated isothermal amplification (LAMP) assay based on the identification of the F1L gene was developed for the specific detection of ORFV infections. The sensitivity and specificity of the LAMP assay were evaluated, and the effectiveness of this method was compared with that of real-time PCR. RESULTS: The sensitivity of this assay was determined to be 10 copies of a standard plasmid. Furthermore, no cross-reactivity was found with either capripox virus or FMDV. The LAMP and real-time PCR assays were both able to detect intracutaneous- and cohabitation-infection samples, with a concordance of 97.83%. LAMP demonstrated a sensitivity of 89.13%. CONCLUSION: The LAMP assay is a highly efficient and practical method for detecting ORFV infection. This LAMP method shows great potential for monitoring the prevalence of orf, and it could prove to be a powerful supplemental tool for current diagnostic methods.

Pathogenic characteristics of three genotype II porcine reproductive and respiratory syndrome viruses isolated from China.

BACKGROUND: We examined differences in pathogenicity in pigs from China that had been experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). METHODS: We compared pathogenic characteristics of a field isolate (GX-1/2008F), two PRRSV isolates (HN-1/2008, YN-1/2008) propagated in cells, and GX-1/2008F that had been propagated in cells (GX-1/2008). The clinical courses, along with humoral and cell-mediated responses, were monitored for 21 days post-infection (DPI). Animals were sacrificed and tissue samples used for gross pathological, histopathological and ultrastructure examination. RESULTS: At 2-3 DPI, animals infected with cell-propagated viruses exhibited signs of coughing, anorexia and fever. However their rectal temperature did not exceed 40.5°C. Viremia was detectable as early as 3 DPI in animals infected with HN-1/2008 and YN-1/2008. Animals inoculated with GX-1/2008F displayed clinical signs at 6 DPI; the rectal temperature of two animals in this group exceeded 41.0°C, with viremia first detected at 7 DPI. Seroconversion for all challenged pigs, except those infected with GX-1/2008, was seen as early as 7 DPI. All of these pigs had fully seroconverted by 11 DPI. All animals challenged with GX-1/2008 remained seronegative until the end of the experiment. Innate immunity was inhibited, with levels of IFN-α and IL-1 not significantly different between control and infected animals. The cytokines IFN-γ and IL-6 transiently increased during acute infection. All virus strains caused gross lesions including multifocal interstitial pneumonia and hyperplasia of lymph nodes. Inflammation of the stomach and small intestine was also observed. Lesions in the group infected with GX-1/2008F were more serious than in other groups. Transmission electron microscopy revealed that alveolar macrophages, plasmacytes and lymphocytes had fractured cytomembranes, and hepatocytes had disrupted organelles and swollen mitochondria. CONCLUSIONS: The pathogenicity of the PRRSV field isolate became attenuated when propagated in MARC-145 cells. Tissue tropism of highly pathogenic strains prevailing in China was altered compared with classical PRRSV strains. The observed damage to immune cells and modulation of cytokine production could be mechanisms that PRRSV employs to evade host immune responses.

Comparative analysis of cloned cDNAs encoding Chinese yellow cattle and Gansu black swine integrin receptors for foot-and-mouth disease virus.

To analyze foot-and-mouth disease virus tropism and host range with respect to the integrin receptor, we cloned cDNAs encoding the integrin αν, ß1, ß3, ß6 and ß8 subunits from Chinese yellow cattle and Gansu black swine and carried out comparative analysis of their molecular characteristics. The lengths of the mature proteins and the functional domains of the four integrin ß subunits were the same between bovine and swine; however, the number of putative N-linked glycosylation sites and cysteine residues and their arrangement varied. Homology analysis of the nucleotide and amino acid sequences showed that FMDV integrin receptors of Chinese yellow cattle and Gansu black swine are highly conserved. Phylogenetic analysis showed that all FMDV integrin receptor subunits of cattle and swine are clustered into the Artiodactyla group; however, Chinese yellow cattle are phylogenetically closer to sheep than to Gansu black swine. We postulate that the host tropism of FMDV may, in part, be related to the divergence of integrin subunits among different species.

Identification of novel B cell epitopes within Toxoplasma gondii GRA1.

Newly synthesized epitopes are one of the most promising antigens for the development of diagnostic kits and peptide vaccines. Very little is known about the B cell epitopes on GRA1 of Toxoplasma gondii, which are recognized by the humoral immune response in pigs. In this study, epitopes derived from GRA1 of T. gondii were identified using synthetic peptide techniques and bioinformatics. Three (PG10, PG13 and PG18) out of the eighteen peptides tested were recognized by all pig sera from different time points after infection, and the other peptides were recognized by select sera from various time points after infection. Our data indicate that many regions of GRA1, and in particular, the regions represented by the peptides PG10, PG13 and PG18, are involved in the pig antibody response. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis.

Immunization with excreted-secreted antigens reduces tissue cyst formation in pigs.

It has been demonstrated that tachyzoite-pooled excreted-secreted antigens (ESAs) of Toxoplasma gondii are highly immunogenic and can be used in vaccine development. However, most of the information regarding protective immunity induced by immunization with ESAs is derived from studies using mouse model systems. These results cannot be extrapolated to pigs due to important differences in the susceptibility and immune response mechanisms between pigs and mice. We show that the immunization of pigs with ESAs emulsified in Freund's adjuvant induced not only a humoral immune response but also a cellular response. The cellular immune response was associated with the production of IFN-γ and IL-4. The humoral immune response was mainly directed against the antigens with molecular masses between 34 and 116 kDa. After intraperitoneal challenge with 10(7) T. gondii of the Gansu Jingtai strain (GJS) of tachyzoites, the immunized pigs remained clinically normal except for a brief low-grade fever (≤40.5 °C), while the control pigs developed clinical signs of toxoplasmosis (cough, anorexia, prostration, and high fever). At necropsy, visible lesions were found at multiple locations (enlarged mesenteric lymph nodes, an enlarged spleen with focal necrosis, and enlarged lungs with miliary or focal necrosis and off-white lesions) in all of the control pigs but not in the pigs that had been immunized. We also found that immunization with ESAs reduced tissue cyst formation in the muscle (P < 0.01). Our data demonstrate that immunization with ESAs can trigger a strong immune response against T. gondii infection in pigs.

Detection of acute toxoplasmosis in pigs using loop-mediated isothermal amplification and quantitative PCR.

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

Small Intestinal Bacterial Overgrowth in Patients with Gallbladder Polyps: A Cross-Sectional Study.

Purpose: There is probably a high prevalence of small intestinal bacterial overgrowth (SIBO) in patients with gallbladder polyps (GBPs). To date, no study has evaluated the occurrence of SIBO in patients with GBPs. The aim of this study was to investigate the prevalence of SIBO in patients with GBPs and explore the possible association between these two conditions. Patients and Methods: The hydrogen-methane breath test was used to diagnose SIBO, and patients were divided into GBPs and control groups based on whether GBPs were found under ultrasound. Clinical and paraclinical factors were compared between the two groups. Results: A total of 297 subjects were included in this study. The prevalence of SIBO was significantly higher in the GBPs group than in the control group (50.0% vs.30.8%, p<0.01). Multivariate logistic regression analysis showed that male (OR=2.26, 95% CI=1.12-4.57, p=0.023), SIBO (OR=3.21, 95% CI=1.69-6.11, p<0.001), fatty liver (OR=2.91, 95% CI= 1.50-5.64, p=0.002) and BMI (OR=1.13, 95% CI=1.01-1.26, p=0.035) were independently associated with GBPs. And by subgroup analysis, we found that the association between SIBO and GBPs was stronger in females than in males (p for interaction< 0.001). In addition, SIBO (OR=5.11, 95% CI=1.42-18.36, p=0.012) and fasting glucose (OR=3.04, 95% CI=1.27-7.28, p=0.013) were found to be associated with solitary polyps. Conclusion: SIBO was highly prevalent in patients with GBPs, and this association seemed to be stronger among females.

Molecular epidemiological investigation of porcine reproductive and respiratory syndrome virus in Northwest China from 2007 to 2010.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease affecting swine worldwide. Northwest China has a sparse pig population and there is no comprehensive information currently available on PRRSV infection. In this study, we analyzed the epidemiological features and genetic diversity of PRRSV from this region. 322 field-isolated tissues or serum samples were collected from aborted pig fetuses or pigs with respiratory disease from 15 herds, twice over a period of 2 years. PRRSV infection was determined and virus strains were classified by the sequencing of GP5. We found that 35.9 % of the animals were PRRSV-positive, and the average prevalence in 2007-2008 and 2009-2010 was 46.5 and 29.3 %, respectively. To further investigate the genetic divergence of PRRSV samples collected from 2007 to 2010, 32 strains were isolated for GP5 sequencing and analysis, and phylogenetic trees were created based on GP5 amino acid sequences. All PRRSVs were of the North American genotype and belonged to the highly pathogenic HP-PRRSV subgenotype. Isolates from the Xinjiang province formed a tightly clustered branch and were closely related to an evolutionary intermediate subgroup isolate. Virus sequences from 2007 to 2008 were compared with those from 2009 to 2010 from the same herd. New mutations were found in isolates after 2009 and focused on nucleotides in the GP5 antibody decoy epitope. PRRSV strains in Northwest China from 2007 to 2010 were similar to those from other regions of China, with some regional characteristics. These results contribute to the knowledge of PRRSV epidemiology in China.

Association between Small Intestinal Bacterial Overgrowth and Subclinical Atheromatous Plaques.

Background: Several recent studies have reported the relationship between atherosclerosis and gut microbial imbalance. Small intestinal bacterial overgrowth (SIBO) is one of the most common forms of gut microbiota imbalance, and studies have shown that SIBO plays an important role in human health. However, the relationship between SIBO and subclinical atheromatous plaques remains unclear. The aim of this study was to investigate the frequency of subclinical atheromatous plaques in patients with SIBO and to explore the association between these two conditions. Methods: A total of 411 eligible subjects were included in this study. The lactulose hydrogen-methane breath test was used to diagnose SIBO, and ultrasound examinations of the carotid, abdominal aorta and lower extremity arteries were performed in all subjects to assess the presence of plaques. Results: Plaques were more common in the SIBO-positive group than in the SIBO-negative group (abdominal aorta, 74.2% vs. 38.8%, p < 0.01; carotid arteries, 71.7% vs. 52.3, p < 0.01; lower extremity arteries, 73.4% vs. 57.6%, p < 0.01). After adjusting for traditional confounders, compared to the SIBO-negative population, the SIBO-positive population had, respectively, OR = 4.18 (95% CI = 2.56−6.80, p < 0.001), OR = 1.93 (95% CI = 1.23−3.02, p = 0.004), OR = 1.81 (95% CI = 1.14−2.88, p = 0.011) and OR = 5.42 (95% CI = 2.78−10.58, p < 0.001) for abdominal, carotid, lower extremity and any-territory plaque presence. Conclusion: SIBO was found to be associated with subclinical atheromatous plaques, and the mechanism of this association warrants further exploration.

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver.

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d(6) as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d(6) were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d(6). The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d(6) was to just compensate the variation of the injections. The detection limit was 5 ng g(-1) swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.

Screening of host genes regulated by ID1 and ID3 proteins during foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

Diagnosis and phylogenetic analysis of Orf virus from goats in China: a case report.

BACKGROUND: Orf virus (ORFV) is the etiological agent of contagious pustular dermatitis and is the prototype of the genus Parapoxvirus (PPV). It causes a severe exanthematous dermatitis that afflicts domestic and wild small ruminants. CASE PRESENTATION: In the present study, an outbreak of proliferative dermatitis in farmed goats. The presence of ORFV in tissue scrapings from the lips was confirmed by B2L gene polymerase chain reaction (PCR) amplification. The molecular characterization of the ORFV was performed using PCR amplification, DNA sequencing and phylogenetic analysis of the B2L gene. CONCLUSION: The results of this investigation indicated that the outbreak was caused by infection with an ORFV that was closely related genetically to Nantou (DQ934351), which was isolated from the Tai wan province of China and Hoping (EU935106), which originated from South Korea in 2008. This is the first report of the phylogenetic analysis of ORFV from goats in China.

Recovery of infectious foot-and-mouth disease virus from full-length genomic cDNA clones using an RNA polymerase I system.

The prototypic foot-and-mouth disease virus (FMDV) was shown more than a century ago to be the first filterable agent capable of causing FMD, and it has served as an important model for studying basic principles of Aphthovirus molecular biology. However, the complex structure and antigenic diversity of FMDV have posed a major obstacle to the attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious FMDV from cDNAs based on an efficient in vivo RNA polymerase I (polI) transcription system. Intracellular transcription of the full-length viral genome from polI-based vectors resulted in efficient formation of infectious virus displaying a genetic marker. Compared with wild-type virus, an abundance of genomic mRNA and elevated expression levels of viral antigens were indicative of the hyperfunction throughout the life-cycle of this cDNA-derived virus at transcription, replication, and translation levels. The technology described here could be an extremely valuable molecular biology tool for studying FMDV complex infectious characteristics. It is an operating platform for studying FMDV functional genomics, molecular mechanism of pathogenicity and variation, and lays a solid foundation for the development of viral chimeras toward the prospect of a genetically engineered vaccine.