Hemagglutinating Encephalomyelitis Virus Research Articles

Development of an Immunochromatographic Strip for Serological Diagnosis of Porcine Hemagglutinating Encephalomyelitis Virus

An immunochromatographic strip was developed for the detection of an antibody against Porcine hemagglutinating encephalomyelitis virus (PHEV). Colloidal gold-labeled rabbit anti-pig immunoglobulin G (IgG) was used as the detection reagent, and the PHEV recombinant antigens and goat anti-rabbit IgG were coated on the prototype strip and the control lines, respectively. The immunochromatographic strip was capable of specifically detecting PHEV antibodies in serum with a hemagglutination inhibition (HI) titer of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Using HI as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 93.41% and 98.42%, respectively. There was a strong agreement between the results obtained by HI and the immunochromatographic strips (κ = 0.926). Additionally, there was a strong agreement between enzyme-linked immunosorbent assay and immunochromatographic strips (κ = 0.929). When the immunochromatographic strip was used for serological diagnosis of 1,117 serum samples in Jilin Province in China, the seropositivity ranged from 6.5% in the Liaoyuan District to 81.6% in the Changchun District. Furthermore, many piglets were seropositive to PHEV, indicating the possible transfer of maternal antibodies via the colostrum. Based on the high specificity, sensitivity, and stability of the immunochromatographic strip, it would be suitable for on-site detection of PHEV antibodies in order to monitor PHEV infections in an animal population.

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

Porcine hemagglutinating encephalomyelitis virus (PHEV) causes vomiting and wasting disease (VWD) or encephalomyelitis, and primarily affects pigs under 3 weeks of age. In this study, we detected PHEV from clinically ill pigs in conventional pig farms in South Korea. From November 2009 to March 2010, a total of 239 pig tissue samples from 91 farms were tested by nested RT-PCR. Among 239 samples, 22 samples from 17 farms were positive for PHEV. The detection rate of suckling pigs, weaning pigs, growers and finishers were 14.3% (12/84), 6.5% (7/107), 7% (3/43), and 0% (0/5), respectively. Symptoms were neurological, respiratory, enteric sign (diarrhea), or nasal bleeding. All pigs were co-infected with other viruses and bacteria and this might have resulted in age variation and clinical signs in the affected pigs. Phylogenetic analysis showed that the PHEV-positive samples and PHEV reference strains were clustered in the same group. These findings imply the presence of only one genogroup of PHEV, regardless of porcine age, clinical signs, and geographical location.

Analysis of the Properties of Neutralizing Monoclonal Antibodies against the Hemagglutinating Encephalomyelitis Virus and Inhibition of HEV Infection by Specific MAb

Eleven monoclonal antibodies (MAbs) that were reactive against the hemagglutinating encephalomyelitis virus (HEV), as seen in the enzyme-linked immunosorbent assay, were obtained. All of these MAbs showed neutralizing activity (1:20,000 to 1:800,000) against the 67N strain of HEV. They also showed hemagglutination inhibition activity (1:400 to 1:409,600). Western blotting tests revealed that all of these 11 MAbs were specific for the S protein of HEV. All MAbs with neutralizing activity showed the same fluorescent staining pattern. Ten-day-old mice pups were immunized with MAb, inoculated with the 10(5) tissue culture-infective dose of HEV at 3 days after immunization, and then examined to determine the viral inhibition. The 1:800-diluted MAb120 inhibited the viral infection in mice pups, though the 1:1,000-diluted MAb120 failed to inhibit the viral infectivity. These MAbs would be a useful tool for rapid and specific diagnosis of HEV and also for antibody-based treatment of the disease.

Propagation of Swine Hemagglutinating Encephalomyelitis Virus and Pseudorabies Virus in Dorsal Root Ganglia Cells

Swine hemagglutinating encephalomyelitis virus (HEV) causes encephalomyelitis or vomiting and wasting disease in suckling piglets. Neurotoropism of the virus has been demonstrated in previous in vivo studies. In the present study, we investigated the infectivity and propagation of HEV in comparison with those of pseudorabies virus (PRV), another neurotropic virus, using dorsal root ganglia cells of newborn mice containing nerve cells and non-neuronal cells. HEV infected nerve cells but did not infect non-neuronal cells, whereas PRV infected both cell types. By using cytoskeletal inhibitors, it was suggested that propagation of HEV and PRV within and among nerve cells depended on microtubules and intermediate filaments of nerve cells, indicating that the viruses may be transported between the cell body and axonal terminals of neurons by fast axonal flow.

Identification of Porcine haemagglutinating encephalomyelitis virus receptor in PK cell membranes

AbstractTo identify Porcine haemagglutinating encephalomyelitis virus (HEV) 67N receptor in porcine kidney (PK) cell membranes, the S1 protein of HEV was expressed in Pichia pastoris and purified by Ni2+ affinity chromatograph. Polyclonal antibodies to HEV were prepared by immunizing rabbits by injecting the purified S1 protein four times. After SDS–polyacrylamide gel electrophoresis (SDS–PAGE), the PK cell membrane proteins were transferred on to nitrocellulose membrane. A virus overlay protein binding assay (VOPBA) was performed using the recombinant S1 protein to identify the protein binding receptor, HEV-S1. The result showed that HEV-S1 protein bound to one band (about 90 kDa) in PK cell membranes. This result is very important for the study of the pathogenic mechanism of HEV.

Genomic characterization of equine coronavirus

The complete genome sequence of the first equine coronavirus (ECoV) isolate, NC99 strain was accomplished by directly sequencing 11 overlapping fragments which were RT–PCR amplified from viral RNA. The ECoV genome is 30,992 nucleotides in length, excluding the polyA tail. Analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (NS2, p4.7, p12.7, and I). The two replicase polyproteins are predicted to be proteolytically processed by three virus-encoded proteases into 16 non-structural proteins (nsp1–16). The ECoV nsp3 protein had considerable amino acid deletions and insertions compared to the nsp3 proteins of bovine coronavirus, human coronavirus OC43, and porcine hemagglutinating encephalomyelitis virus, three group 2 coronaviruses phylogenetically most closely related to ECoV. The structure of subgenomic mRNAs was analyzed by Northern blot analysis and sequencing of the leader–body junction in each sg mRNA.

Analysis of the genome sequence of an alpaca coronavirus

Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3′ end. This virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs) characteristic of Group 2 bovine coronavirus (BCoV). Phylogenetic analysis showed that the ACoV genome is clustered closely (> 99.5% identity) with two BCoV strains, ENT and LUN, and was also closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (> 96%), and porcine hemagglutinating encephalomyelitis virus (> 93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT, respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same ancestor as BCoV ENT and LUN.

The morphogenesis and trans-neuronal transfer of swine hemagglutinating encephalomyelitis virus in the central nervous system

D t s K K parable to those we use today, that all our joys and fears, prejudices and desires, emanate from actions in the brain. This is in striking contrast to contemporary Egyptian usagewhere, in punctilious funereal practice, the brain was heedlessly discarded. More than 2000 years ensued, however, before it was recognized that the mind derived from “gray matter” rather than the “air” (pneuma) in the cerebral ventricles. Abstracts / Neuroscience

Evolutionary History of the Closely Related Group 2 Coronaviruses: Porcine Hemagglutinating Encephalomyelitis Virus, Bovine Coronavirus, and Human Coronavirus OC43

The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.

Neurotropism of Swine Haemagglutinating Encephalomyelitis Virus (Coronavirus) in Mice Depending upon Host Age and Route of Infection

Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10 7PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules

5′ and 3′ UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5′ UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5′ UTR and adjacent 289-nt sequences, and 3′ UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3′ UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5′ and 3′ replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.

Sequence of the 3′-terminal end (8·1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses

A cytopathogenic coronavirus, serologically identified as porcine haemagglutinating encephalomyelitis virus (HEV), has recently been associated with acute outbreaks of wasting and encephalitis in nursing piglets from pig farms in southern Québec and Ontario, Canada. The 3'-terminal end of the genome of the prototype HEV-67N strain and that of the recent Québec IAF-404 field isolate, both propagated in HRT-18 cells, were sequenced. Overall, sequencing data indicated that HEV has remained antigenically and genetically stable since its first isolation in North America in 1962. Compared with the prototype strain of bovine enteropathogenic coronavirus (BCoV), HEV, as well as the human respiratory coronavirus (HCoV-OC43) showed a major deletion in their ORF4 gene. Deduced amino acid sequences for both HEV strains revealed 89/88, 80, 93/92 and 95/94% identities with the structural proteins HE, S, M and N of BCoV and HCoV-OC43, respectively. Major variations were observed in the S1 portion of the S gene of both HEV strains, with only 73/71% amino acid identities compared with those of the two other haemagglutinating coronaviruses.

Susceptibility of Rats of Different Ages to Inoculation with Swine Haemagglutinating Encephalomyelitis Virus (a Coronavirus) by Various Routes

Haemagglutinating encephalomyelitis virus, strain 67N, was used to inoculate 1-, 2-, 4- and 8-week-old rats by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) and oral routes with graded doses. The routes of infection, in descending order of efficacy, were: i.ci.ns.ci.pi.v. and oral. Rats aged 1 and 2 weeks were generally similar in terms of mortality and mean time to death, regardless of inoculation route, except for the oral route, which had little effect. In comparison with the 1- and 2-week-old rats, the 4-week-old rats were less susceptible to the virus by all routes. Eight-week-old rats inoculated by the i.ci.n. or s.c. routes died, but all those inoculated by other routes survived. To follow the spread of virus in the central nervous system, 4-week-old rats inoculated by the i.c. route were examined. The virus was first detected in the brain on day 1 and in the spinal cord on day 2. The viral titres in both tissues reached a plateau of 107plaque-forming units (PFU)/0·2 g by day 4, at which time clinical signs had developed. By immunohistochemical analysis, virus-specific antigen was found first in the pyramidal cells of the hippocampus and cerebral cortex, and later in the large-sized neurons of the pons and spinal cord. Still later (day 4) immunolabelling was found in Purkinje cells of the cerebellum, but not in the ependymal cells, choroid plexus or other glial cells.

Infection of hemagglutinating encephalomyelitis virus (HEV) at the visual pathways of rats.

Hemagglutinating encephalomyelitis virus (HEV) is neurotropic Coronavirus causing vomiting and wasting disease or encephalitis in piglets (Andries and Pensaert. 1981). In the infected animals, the virus reaches the CNS through the nerve pathways from peripheral nerve. Our previous studies have demonstrated that HEV propagated through nervous route and its infection was restricted to neurons after inoculation into sciatic nerve or footpad of rats (Hirano et al., 1995, 1998). Our findings suggest that HEV is useful as a trans-synaptic tracer for analyzing neuronal connections in the CNS. The present study was performed to examine and evaluate the usefulness of HEV as a trans-synaptic tracer in the visual pathway.

Biological and molecular characteristics of an HEV isolate associated with recent acute outbreaks of encephalomyelitis in Quebec pig farms.

Porcine hemagglutinating encephalomyelitis virus (HEV) is a member of the antigenic subgroup of hemagglutinating coronaviruses, including human respiratory coronavirus HCV-OC43, bovine and turkey coronaviruses, and mouse hepatitis virus (Spaan et al., 1988). These viruses share antigenic determinants located on their homologous structural proteins N, M, HE and S. The HEV specifically infects swine, having a strong tropism for epithelial cells of the upper respiratory tract and for the central nervous system (CNS) (Andries and Pensaert, 1980). The virus causes two clinical syndromes: the vomiting and wasting disease (VWD) and non-suppurative encephalomyelitis (Pensaert and Andries, 1993). The mode of transmission is through nasal secretions and the virus spread via the peripheral nerves to the CNS. The HEV infection is believed to be widespread in many pig producing countries as a subclinical condition.

Spread of hemagglutinating encephalomyelitis virus (HEV) in the CNS of rats inoculated by intranasal route.

HEV is known to cause vomiting and wasting disease or encephalomyelitis in piglets (Andries et al., 1980,1981; Mengeling et al., 1972; Roe et al., 1958). In experimental oronasal infection of piglets, the virus spread to the CNS predominantly via the nerve pathways (Andries et al., 1980, 1981). In our previous study, 4-week-old rats died of encephalitis after inoculation by different routes including intranasal route (Hirano et al., 1993). In the infected animals, the virus reaches the CNS through the nerve pathways from peripheral nerve. Our previous studies have demonstrated that HEV propagated through nervous route and its infection was restricted to neurons after inoculation into sciatic nerve or footpad of rats (Hirano et al., 1995, 1998). Our findings suggest that HEV is useful as a trans-synaptic tracer for analyzing neuronal connections in the CNS.

Characterization of a coronavirus isolated from a diarrheic foal.

A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.

Pigs with highly prevalent antibodies to human coronavirus and swine haemagglutinating encephalomyelitis virus in the Tohoku District of Japan.

From 1985 to 1988, a total of 2496 swine sera from 60 farms in the Tohoku District of the Honshu Island of Japan were examined for antibodies to swine haemagglutinating encephalomyelitis virus (HEV), human coronavirus (HCV) and bovine coronavirus (BCV) by haemagglutination-inhibition (HI) test. Antibodies to HEV 67N strain and HCV OC43 strain were highly prevalent with positivity rates of 82.1 and 91.4%, respectively, while seropositivity rate to BCV Kakegawa strain was 44.2%. No clinical signs of HEV infection were noticed in any farms including farms with relatively high seropositivity. The results suggested that HCV or antigenitically related virus(es) as well as HEV might be perpetuated in swine in the Tohoku District.

A serological survey of human coronavirus in pigs of the Tohoku District of Japan.

A total of 2496 swine sera from 60 farms in the Tohoku District of Japan was examined for hemagglutination inhibiting (HI) antibodies to human coronavirus (HCV), swine hemagglutinating encephalomyelitis virus (HEV) and bovine coronavirus (BCV). HI antibodies to HCV OC43 strain and HEV 67N strain were highly prevalent with positivity rates of 91.4 and 82.1%, respectively, while the BCV Kakegawa strain was 44.2% positive. Farm D in Miyagi Prefecture showed the highest antibody titers to HCV OC43 strain with geometric mean titers (GMT) of 1:200. These results suggest that pigs might be infected with HCV or an antigenetically related virus as well as HEV.

Spread of swine hemagglutinating encephalomyelitis virus from peripheral nerves to the CNS.

Swine hemagglutinating encephalomyelitis virus (HEV) strain 67N was inoculated into the sciatic nerve or the right leg crural muscle of rats. In both cases, the virus was isolated first from the caudal half of the spinal cord on day 2 after inoculation, and from the rostral half of the spinal cord and the brain on day 3. The virus titers in the brain reached a maximum when the infected rats developed CNS symptoms on day 5. Using confocal laser scanning microscope, fluorescent positive cells were first found in the lumbar dorsal root ganglion (DRG) and spinal cord ipsilateral of the inoculated leg on day 3. Antigen positive neurons were found bilaterally in the lumbar DRG and spinal cord on day 4. On day 5 specific fluorescence was observed in the neurons of the cerebral cortex, hippocampus, brainstem and Purkinje cells in the cerebellum.