Development of Reverse Transcriptase PCR and Nested PCR to Detect Porcine Hemagglutinating Encephalomyelitis Virus

Abstract

ABSTRACT. Porcine hemagglutinating encephalomyelitis virus (HEV) causes encephalomyelitis, or vomiting and wasting disease, in suckling piglets. The mortality rate for piglets under 3 weeks old is 100%, but they are usually protected by maternal antibodies. Recently, the risk of an HEV outbreak has increased in the pig industry, because of widely using specific pathogen-free pigs that have no antibodies to HEV. We developed reverse transcription (RT) PCR and nested PCR to detect HEV. Primer sets of polymerase, non-structural protein, and spike protein were designed for RT-PCR and nested PCR based on the nucleotide sequences of the HEV 67N strain. The PC R designated primer sets of spike protein detected only HEV viral RNA among other related nidoviruses. Detection of HEV viral RNA by nested PCR was more sensitive than virus isolation in cell cultures. Nested PCR detected HEV viral RNA from experimentally infected samples of mice and field samples of piglets. The RT-PCR and nested PCR methods to detect HEV is considered a good way to show the HEV etiology on pig farms. KEY WORDS: HEV, nested PCR, RT-PCR.

Detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in South Korea

“…In Korea, there has not been a serious outbreak of PHEV, which could cause high mortality in young piglets. However, outbreaks occurred in other countries; 2001 in Japan [24], 2002 in Canada [1], and 2006 in Argentina [17]. This might imply that PHEV is a reemerging infection that could well be the next important porcine disease virus.…”

“…However, when PHEV-seronegative piglets (\3 weeks old) are introduced to this virus, the mortality reaches almost 100%. This is significant for gnotobiotic pig farms or SPF pig farms [24] where the pigs do not receive colostral antibodies. Also, even in not one of those specialized farms, nonimmune subpopulations that might exist in a large gilt pool could act as potential sources of PHEV [17] to induce clinical syndromes, the vomiting and wasting disease (VWD) and/or the encephalomyelitis.…”

“…For serological studies, hemagglutination inhibition (HI) test was used in most cases and virus neutralization (VN) test has been also available for detection of PHEV antibody [20,22]. For more rapid and accurate PHEV diagnosis, without the need for virus strain, reverse transcriptase PCR and nested PCR methods were developed to detect PHEV using spike gene specific primers [24]. The spike gene of PHEV appears to be the most variable region among coronaviruses [21,25], which can be exploited to enhance the specificity of PHEV detection.…”

Attempted Experimental Reproduction of Porcine Periweaning-Failure-to-Thrive Syndrome Using Tissue Homogenates

“…The filtered and non-filtered homogenates were tested for PCV2 [7], PRRSV (Tetracore EZ-PRRSV™ Kit; Rockville, MD, USA), influenza A virus [8], group A rotavirus (unpublished data), HEV [9], TGE [10], PEV_1, 2, and 3 [11], PCMV [12], PECV [13], Helicobacter-pylori -like organism and Helicobacter-heilmannii -like organism [14] and Mhyo [15] by PCR.…”

Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus

“…At present, various laboratory methods are available for the detection and surveillance of PHE-CoV, including virus isolation [1], hemagglutination/hemagglutination inhibition (HA/HI) tests [13], immunohistochemistry (IHC) assays [10], and molecular tools such as nestedpolymerase chain reaction (nested PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) that enable detection of specific CoV RNA sequences from infected tissues [14,15]. However, these detection methods are laborious, time-consuming, and require laboratory procedures or special equipment, making them unsuitable for on-site inspection.…”