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.

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.