A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/µl for ASFV, PCV2 and PRRSV, 100 copies/µl for SVDV, CSFV, VESV and 1,000 copies/µl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Bile acids promote the expression of hepatitis C virus in replicon-harboring cells.

Hepatitis C virus (HCV) is a cause of chronic liver disease, with more than 170 million persistently infected individuals worldwide. Although the combination therapy of alpha interferon (IFN-alpha) and ribavirin is effective for chronic HCV infection, around half of all patients infected with HCV genotype 1 fail to show sustained virologic responses and remain chronically infected. Previously, we demonstrated that bile acids were essential for growth of porcine enteric calicivirus in cell culture in association with down-regulation of IFN responses. Because hepatocytes are exposed to high concentrations of bile acids in the liver, we hypothesized that bile acids have similar effects on HCV replication. We incubated HCV replicon-harboring cells (genotype 1b, Con1) in the presence of various bile acids and monitored the expression of HCV RNA and protein (NS5B). The addition of an individual bile acid (deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, or glycochenodeoxycholic acid) in the medium increased the levels of HCV RNA and proteins up to fivefold at 48 h of incubation. An antagonist of bile acid receptor farnesoid X receptor (FXR), Z-guggulsterone, reduced the bile acid-mediated increase of HCV RNA. When IFN (alpha or gamma) and each bile acid were incubated together, we observed that bile acid significantly reduced the anti-HCV effect of IFN. These results indicated that bile acids are factors in the failure of IFN treatment for certain patients infected with HCV genotype 1. Our finding may also contribute to the establishment of better regimens for treatment of chronic HCV infections by including agents altering the bile acid-mediated FXR pathway.

X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity.

Caliciviruses, grouped into four genera, are important human and veterinary pathogens with a potential for zoonosis. In these viruses, capsid-related functions such as assembly, antigenicity, and receptor interactions are predominantly encoded in a single protein that forms an icosahedral capsid. Understanding of the immunologic functions and pathogenesis of human caliciviruses in the Norovirus and Sapovirus genera is hampered by the lack of a cell culture system or animal models. Much of our understanding of these viruses, including the structure, has depended on recombinant capsids. Here we report the atomic structure of a native calicivirus from the Vesivirus genus that exhibits a broad host range possibly including humans and map immunological function onto a calicivirus structure. The vesivirus structure, despite a similar architectural design as seen in the recombinant norovirus capsid, exhibits novel features and indicates how the unique modular organization of the capsid protein with interdomain flexibility, similar to an antibody structure with a hinge and an elbow, integrates capsid-related functions and facilitates strain diversity in caliciviruses. The internally located N-terminal arm participates in a novel network of interactions through domain swapping to assist the assembly of the shell domain into an icosahedral scaffold, from which the protruding domain emanates. Neutralization epitopes localize to three hypervariable loops in the distal portion of the protruding domain surrounding a region that exhibits host-specific conservation. These observations suggest a mechanism for antigenic diversity and host specificity in caliciviruses and provide a structural framework for vaccine development.

Crystallization and preliminary crystallographic analysis of San Miguel sea lion virus: an animal calicivirus.

The Caliciviridae is a family of nonenveloped, icosahedral, positive-sense single-stranded RNA viruses. This family of viruses consists of both animal and human pathogens. Adapting human caliciviruses to cell culture has not been successful, whereas some animal caliciviruses, including San Miguel sea lion virus, have been successfully propagated in vitro. Here we report the crystallization of San Miguel sea lion virus serotype 4 (SMSV4) and the preliminary X-ray crystallographic analysis of the crystals. SMSV4 have been crystallized using the hanging-drop method. These crystals diffracted to approximately 3A resolution using a synchrotron radiation source. A single crystal under cryo-conditions yielded a complete set of diffraction data. Data processing of the diffraction patterns showed that SMSV crystals belong to I23 space group with cell dimensions a=b=c=457 A. The crystallographic asymmetric unit includes five icosahedral asymmetric units, each consisting of three capsid protein subunits. In the space group I23, given the icosahedral symmetry and the size of the virus particle, the location of the particle is constrained to be at the point where the crystallographic 2- and 3-fold axes intersect. The orientation of the virus particle in the unit cell was ascertained by self-rotation function calculations.

Development of a reverse transcription polymerase chain reaction procedure for the detection of marine caliciviruses with potential application for nucleotide sequencing.

A reverse transcription polymerase chain reaction (RT-PCR) procedure is described for the detection of marine caliciviruses including vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), bovine Tillamook virus (BCV Bos-1) and caliciviruses (CV) isolated from dolphin (Cetacean CV), gorilla (Primate CV) and rattlesnake (Reptile CV) using primers (1F and 1R) designed from the capsid-coding region of the viral genome. These primers were compared with those described by Neill, J.D. and Seal, B.S., 1995: Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea lion and vesicular exanthema of swine viruses, Mol. Cell. Probes 9, 33-38 (Hel1/Hel2), which had been designed from the 2C-like region of the calicivirus genome. Both sets proved to be extremely useful diagnostic tools for all of the known marine calicivirus serotypes with the exception of three: SMSV-8 and -12 and mink CV suggesting that these three caliciviruses may belong to a different group. Neither of the two primer sets reacted with strains of the vesicular disease viruses of foot-and-mouth disease (FMD), swine vesicular disease (SVD) or vesicular stomatitis (VS) nor with two feline caliciviruses (FCV). The 1F/1R primer set has the advantage over the Hel1/Hel2 set in that it generates a larger PCR product for nucleotide sequence investigations and so provides greater opportunity for identifying molecular differences between the viruses.

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus.

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.

The capsid protein of vesicular exanthema of swine virus serotype A48: relationship to the capsid protein of other animal caliciviruses.

Vesicular exanthema of swine virus (VESV), the prototype calicivirus, is the etiologic agent of the porcine disease vesicular exanthema of swine (VES). VES is characterized by vesicle formation on the extremities, mouth and snout and causes abortions and stillbirths if infection occurs during pregnancy. VESV is considered an exotic agent in the US, following its eradication in 1956. The single capsid protein gene of VESV serotype A48 was cloned and sequenced. The capsid amino acid sequence was 69% similar to the San Miguel sea lion virus serotype 1 (SMSV 1) and 89% similar to the SMSV serotype 4 (SMSV 4) capsid proteins. The six functional regions (A-F) previously identified in SMSV 1, SMSV 4, feline calicivirus and rabbit hemorrhagic disease virus capsid proteins were present in VESV A48. Two sets of PCR primers were designed which directed amplification of the 5' end (A region) and the hypervariable (E region) sequences of the capsid protein precursor gene of these viruses, as well as seven additional SMSV serotypes. Alignment and phylogenetic analysis of the N-terminal sequences demonstrated the close relationship of these viruses. Alignment of the hypervariable region amino acid sequences of the ten viruses confirmed that a great variety of sequence exists in this region; however, a consensus sequence (NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R)) was derived which is also present in the feline calicivirus capsid protein. Comparison of the E region sequences provides further evidence that this area of animal calicivirus capsid protein may contain the major antigenic determinants.

Viral serologic survey of bowhead whales in Alaska.

Serum samples from 21 of 36 Eskimo harvested bowhead whales (Balaena mysticetus) were positive by virus neutralization (50% endpoint titer > or = 1:28 and/or 100% endpoint titer > or = 1:20) for antibodies to at least one virus serotype from the calicivirus family, vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV). Many animals were positive to more than one serotype when using the Spearman-Karber (S-K) method for calculating antibody titers. The most common serotype detected was VESV F55 with 6 of 36 (17%) by the Monto and Bryan (MB) titer calculation method, and 17 of 36 (47%) by the S-K titer calculation method. Vesicular exanthema of swine virus 1934B antibody was detected in 3 of 36 (8%) and 5 of 36 (14%) whales using the MB and S-K methods, respectively. Vesicular exanthema of swine virus J56 antibody was detected in 3 of 36 (8%) by the S-K method only. All whales < 8.5 m (estimated yearlings, n = 6) were seronegative for VESV J56 and 1934B while 10% and 17% of the whales > 8.5 m were positive, respectively. Whales assumed to be sexually mature (> 13 m) had a higher prevalence of antibody to VESV 1934B and SMSV 8 than those < 13 m. Gender had an effect on seroprevalence of antibody to VESV 1934B as titers > or = 1:28 (S-K method) occurred in 18% of the females and 7% of the males. Antibody to other serotypes (SMSV 8 and 12) occurred less frequently (< 6%) at an antibody titer > or = 1:28 by the S-K method. All 36 whale sera were negative for antibody to VESV-A48, B51, C52, D53, E54, G55, H54, I55, and K54; Tillamook calicivirus, and dolphin morbillivirus; and SMSV-1, 2, 4, 5, 6, 7, 9, 10, 11, and 13 by the S-K method.

Detection of early infection of swine vesicular disease virus in porcine cells and skin sections. A comparison of immunohistochemistry and in-situ hybridization.

Sensitive methods are required to study the early pathogenesis of swine vesicular diseases (SVD). Therefore, two new methods, immunohistochemistry (IHC) and in-situ hybridization (ISH), were developed and tested for their specificity and sensitivity. With these methods the SVD virus (SVDV) infection in cytospins of primary porcine kidney cells and in frozen skin sections was investigated. Both IHC and the ISH showed a specific cytoplasmic staining, but the IHC detected more infected cells than the ISH. Furthermore, both IHC and ISH were able to detect SVDV in skin sections 4.5 h after infection. It is concluded that IHC is the most suitable and simplest method to identify cells and tissues that support the initial replication of swine vesicular disease virus. However, IHC can only be applied to frozen sections, whereas ISH can also be used in paraformaldehyde-fixed tissues.

Genetic relatedness of the caliciviruses: San Miguel sea lion and vesicular exanthema of swine viruses constitute a single genotype within the Caliciviridae.

The San Miguel sea lion viruses (SMSV) and vesicular exanthema of swine viruses (VESV) are related morphologically and antigenically, but little has been done to determine their genotypic relationship to each other and to other caliciviruses. To examine this relationship, reverse transcriptase PCRs were performed by using oligonucleotide primer sets designed to amplify portions of the 2C RNA helicase-like and RNA-dependent RNA polymerase regions with total cellular RNA purified from virus-infected cell cultures as a template. The 2C RNA helicase primers directed the amplification of this region from eight SMSV serotypes, five VESV serotypes, and four related viruses. The RNA polymerase primer sets amplified products from all these viruses except one. Phylogenetic comparison of the caliciviruses demonstrated that SMSV, VESV, and four related viruses are closely related while being distinct from feline calicivirus, the human caliciviruses (small, round-structured viruses), and rabbit hemorrhagic disease virus and that they should be classified as a single genotype within the Caliciviridae.

Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea-lion and vesicular exanthema of swine viruses.

The San Miguel sea-lion viruses (SMSV) and vesicular exanthema of swine viruses (VESV) are members of the calicivirus family and aetiologic agents of vesicular disease in susceptible hosts. These two virus groups have been shown by several serological methods to be closely related antigenically. To further examine their relatedness, two sets of non-degenerate oligonucleotide primers were designed for the specific amplification of two distinct regions of the SMSV and VESV genomes using a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol. The sequence of the primers were based on the nucleotide sequence of SMSV serotypes 1 and 4. The RNAs from a number of SMSV serotypes and a single VESV isolate were used as template in this study. These included SMSV serotypes 1, 2, 4, 5, 6, 7, 13 and 14 and VESV serotype A48. Also included in this study were Tillamook calicivirus (Bos-1 calicivirus, BCV) and a recently isolated skunk calicivirus (SCV). The first primer set amplified a 357-bp fragment from the 2C-like or RNA-helicase-encoding region (11 of 11 viruses) and the second set amplified a fragment from the RNA-dependent RNA polymerase region (520 bp, 9 of 11 viruses). These primer sets did not amplify product from either feline calicivirus or mink calicivirus. The results of this study demonstrate the genetic relatedness of SMSV and VESV and the potential usefulness of RT-PCR to detect and identify these viruses in diagnostic and routine screening applications.

Prevalence and distribution of four serotypes of SMSV serum neutralizing antibodies in wild animal populations.

Serum neutralizing antibodies to four serotypes of San Miguel Sea Lion Virus (SMSV) were demonstrated in a variety of marine and terrestrial species. These results show a wide geographic distribution of SMS viruses in the marine environment and indicate that certain terrestrial mammals have been infected with these so-called marine viruses. Evidence is presented supporting the theory that unidentified submammalian marine species are a reservoir for SMSV.