Virus isolated and immunofluorescence in different organs of pigs infected with hemagglutinating encephalomyelitis virus

Hemagglutinating encephalomyelitis virus (HEV; also designated vomiting and wasting disease virus) was inoculated oronasally in 26 colostrum-deprived pigs. Anorexia and vomition were seen after an incubation period of 4 to 6 days. In pigs killed during the incubation period or within 2 days after the onset of the clinical signs, HEV could be isolated regularly from the tonsils and the respiratory tract, irregularly from the digestive tract, rarely from the blood, and never from lymph nodes and spleen. The brainstem almost always contained virus after clinical signs appeared, but was only one positive during the incubation period. Olfactory bulb, cerebrum, cerebellum, and vagal nerve were also frequently virus positive in pigs which were ill when killed. The results of the examination by immunofluorescent antibody technique indicated that HEV multiplies in the epithelium lining the respiratory tract and the tonsillar crypts, in neuroepithelium of the nasal mucosa, and in neurons of the digestive tract. The neuronotropism of HEV was also shown by the presence of fluorescence in the perikaryon of neurons in the brainstem and in the trigeminal ganglion without the involvement of other cell types. The presence of viral antigens in the perikaryon of trigeminal sensory ganglion cells in pigs killed during the incubation period was considered as positive evidence for viral spread via nerves.     

The vagus nerve is one route of transneural invasion for intranasally inoculated influenza a virus in mice

Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem. Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. To evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b) and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. An asymmetric distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side 2 days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The results of this study, thus, demonstrate that neurotropic influenza virus travels to the CNS mainly via the vagus nerve.     

Investigation of pseudorabies virus latency in nervous tissues of seropositive pigs exposed to field strain

The prevalence and quantity of latent pseudorabies virus (PrV) in nervous tissues of pigs exposed to field strain in Korea was investigated by nested and real-time PCR. Nervous tissues including trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS) were collected from 94 seropositive pigs. PrV latent infection in nervous tissues was initially investigated by nested PCR targeting three glycoprotein genes (gB, gE, and gG). Based on the obtained result, latent infection was detected in 95.7% of screened animals. Furthermore, it was revealed that the examined tissues harbored different copy numbers of latent PrV genome ranging from <10(2.0) to 10(7.1) copies per microgram of genomic DNA in real-time PCR analysis. These results show that under normal conditions, levels of latent PrV in the nervous tissues of pigs can vary across a wide range. Therefore, the data presented here provides information regarding control of the endemic state of PrV in Korea.     

Immunohistochemical analysis of pseudorabies virus spread through neurons innervating the eyeball

Pseudorabies virus (PRV) was inoculated intraocularly into BALB/c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina; all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread.     

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.     

Neurotropism of the 1997 Hong Kong H5N1 influenza virus in mice

The direct transmission of H5N1 influenza A viruses from chickens to humans in Hong Kong in 1997 emphasized the need to have information on the pathogenesis of avian influenza virus infection in mammals. H5N1 influenza viruses isolated from patients during the incident killed experimentally infected mice. The principal lesions of the mice were broncho-interstitial pneumonia and nonsuppurative encephalitis. Infectious viruses and/or viral antigens were detected in the brain as well as in the trigeminal and vagal ganglia but not in the blood of the mice. These findings suggest that the virus reached the brain through the vagus and/or trigeminal nerves following replication in the respiratory mucosa. The results imply that neurotropism of the H5N1 virus in mice is a novel characteristic in the pathogenesis of infection by human influenza virus isolates.     

Neural invasion of two virulent suid herpesvirus 1 strains in neonatal pigs with or without maternal immunity.

The neural invasion of two virulent Suid Herpesvirus 1 (SHV1) strains was examined in neonatal pigs with or without maternal immunity. One-week-old pigs with comparable levels of maternal immunity (SN-titer = 12-48) were intranasally inoculated with 10(7.0) TCID50 of either of the Ka or E21 strains. The invasion of the strains was examined in the nasal mucosa and in three neuronal levels of the trigeminal nervous pathway as well as in three levels of the olfactory nervous pathway by virus titration and immunohistochemistry (IHC). In control pigs without specific antibodies, both strains invaded up to the end level of each neural pathway. In pigs with maternal immunity, the Ka strain invaded only up to the 2nd level of each pathway with titers being significantly lower (p<0.05) than in the negative controls. However, the E21 strain invaded up to the end levels in both neural pathways of immune pigs with virus titers being similar to those observed in non-immune pigs (p>0.05). IHC revealed that maternal antibodies can protect against a fibroblast-mediated spread of the Ka strain in the lamina propria of the nasal mucosa, as well as against a local spread of the Ka and E21 strains from neurons to their satellite cells in the trigeminal ganglion. In conclusion, the nature of virus strain determines the invasion of SHV1 within the nervous system of maternally-immune neonatal pigs.     

Viral antigen distribution in the central nervous system of cattle persistently infected with bovine viral diarrhea virus

Distribution of viral antigens in the central nervous system of 25 cattle with a persistent bovine viral diarrhea virus (BVDV) infection was studied. Using a polyclonal antiserum produced in pigs and the direct immunofluorescence and immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus; in other areas of brain and spinal cord, viral antigens were in single neurons or small groups of neurons. There was no morphological evidence of cellular alteration due to viral persistence. Perivascular lymphocytic infiltrations were in affected nervous tissue. It is concluded that the central nervous system is an important location for persistence of BVDV.     

Invasion and spread of single glycoprotein deleted mutants of Aujeszky''s disease virus (ADV) in the trigeminal nervous pathway of pigs after intranasal inoculation

The purpose of the study was to evaluate the role which non-essential envelope glycoproteins play in the neuroinvasion and neural spread of ADV. The invasion and spread in the trigeminal nervous pathway with the Ka strain of ADV and its single deletion mutants Ka gI⁻, Ka gp63⁻ and Ka gIII⁻ were examined after intranasal inoculation in neonatal pigs by virus isolation and immunocytochemistry. Evaluation was performed in the nasal mucosa, trigeminal ganglion (1st neuronal level), pons-medulla (2nd neuronal level) and thalamus-cerebellum (3rd neuronal level). The Ka gIII⁻ mutant invaded up to the 3rd neuronal level of the trigeminal pathway and spread in a similar way to the parental Ka strain. The Ka gp63⁻ mutant invaded up to the 3rd neuronal level but the spread of this mutant was impaired at all the neuronal levels. The Ka gI⁻ mutant was least neuroinvasive and reached only up to the 2nd neuronal level. The results showed that glycoproteins gI and gp63 play a role in the invasion and spread of ADV in the nervous system. However, the gI glycoprotein appears to be the most important for neuroinvasion and neural spread of ADV in pigs. Therefore, gI deleted vaccines may be considered to be safer with respect to the neuroinvasion than vaccines carrying single deletions of other non-essential envelope glycoproteins.     

Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax)

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.     

Investigation of sites of pseudorabies virus latency, using polymerase chain reaction.

Pseudorabies virus (PRV) latency was investigated, using polymerase chain reaction (PCR). A PCR protocol was developed that specifically amplified a 217-base pair sequence within the gene encoding the essential glycoprotein gp50 of PRV. Using this PCR procedure, the gp50 sequence was amplified from tissues of pigs infected with various doses of PRV (Becker strain). At postinoculation day 64, viral isolation was performed on nasal swab specimens and homogenates of tonsils and trigeminal nerve ganglia obtained from 11 PRV-convalescent, seropositive pigs. Results were negative in all cases. By use of PCR, 11 of 11 pigs had PRV-positive trigeminal nerve ganglia and brain stem, 10 of 11 pigs had PRV-positive tonsils, and 9 of 11 pigs had PRV-positive olfactory bulbs.     

Immunohistochemical detection of porcine teschovirus antigen in the formalin-fixed paraffin-embedded specimens from pigs experimentally infected with porcine teschovirus

Porcine teschovirus (PTV) antigens were detected by a streptavidin-biotin complex method in formalin-fixed paraffin-embedded tissues of 3-week-old pigs that had been inoculated intravenously with PTV Talfan strain. PTV antigens were detected in cytoplasm of nerve cells, glial cells and endothelial cells in the cerebellar nuclei, the grey matter of the midbrain, pons and medulla oblongata and the ventral horn of the spinal cord and of ganglion cells in the spinal ganglion corresponding to those lesions characterized as non-suppurative encephalomyelitis and ganglionitis. The results of this study suggest that nerve cells of the brain stem and spinal cord and ganglion cells of the spinal ganglion permit PTV replication and represent the main target cell population of PTV. This is the first study to demonstrate PTV antigen by immunohistochemistry in formalin-fixed paraffin-embedded tissue specimens from pigs infected with PTV.     

Neural changes in recurrent infection of infectious bovine rhinotracheitis virus in calves treated with dexamethasone.

Recurrent infection by infectious bovine rhinotracheitis (IBR) virus was induced in calves by dexamethasone (DM) treatment (given 5 days) at 5 months after primary infection. The virus appeared in nasal secretions of the calves on the 4th day after initiation of DM treatment and continued until the 9th day. The calves were killed on the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, and 11th days after DM treatment was started for examination by histopathologic and immunofluorescent antibody techniques. The most significant neural change was trigeminal ganglionitis with neuronophagia, which was observed from the 3rd to the 11th day. Significantly, the extent of changes in the trigeminal ganglion and medulla oblongata corresponded to the amount of DM treatment administered. The IBR virus antigen was first observed in the trigeminal ganglion cells, and thereafter, it was detected in the Schwann cells, satellite cells, neuroglia cells, and nasal mucosa until the 10th day. These observations indicate that the IBR virus is capalbe of producing a persistent infection in the trigeminal ganglion and that trigeminal ganglionitis may be a characteristic lesion for inducing the reactivation of lagent IBR virus.     

Experimental Aujeszky's disease in pigs: excretion, survival and transmission of the virus

Airborne Aujeszky's disease virus was recovered from looseboxes containing groups of pigs infected with virus strains from England, Northern Ireland and Denmark from days 1 to 7 after infection. Pigs sampled individually excreted most airborne virus on days 2 and 3 after infection. On a 24 hour basis the maximum amount of airborne virus excreted per pig was log10 5.3 TCID50. Subclinical infection was transmitted from a clinically affected group of pigs to a seronegative group held in separate looseboxes when air was drawn through ducting connecting one box with the other. Tissues taken from pigs killed at varying times after infection showed that the main sites of virus replication were in the head and neck region. Aujeszky's disease virus was detected for up to 40 days in a range of tissues taken from pigs at the acute stage of disease and stored at -20 degrees C.     

Pseudorabies virus induces a rapid up-regulation of nitric oxide synthases in the nervous system of swine

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.     

Experimental latent and recrudescent bovine herpesvirus-1 infections in calves

Latent bovine herpesvirus-1 (BHV-1) infection was established in 6 calves and was demonstrated by reinduction of virus shedding after administration of corticosteroids. Latently infected calves failed to transmit BHV-1 during 4 weeks' contact with sentinel calves. Infected calves were killed and necropsied during latency or induced recrudescence. The BHV-1 DNA was demonstrated intranuclearly in trigeminal ganglion neurons by in situ hybridization. The BHV-1 antigen was demonstrated by immunofluorescence in trigeminal ganglion neurons during recrudescence. By electron microscopy, changes in the appearance of the Nissl bodies and a high frequency of nuclear bodies were observed in trigeminal ganglion neurons.     

Feline viral rhinotracheitis virus: explant and cocultivation studies on tissues collected from persistently infected cats

Tissues from 16 feline viral rhinotracheitis (FVR) carrier cats and 15 controls were examined by the techniques of homogenisation, cocultivation and explant cultivation. The tissues examined included trigeminal ganglion and maxillary nerve, olfactory lobe and nerve endings, tonsils, submandibular lymph node, spleen and parotid salivary gland. Most of the cats were shedding FVR virus in the oropharynx at the time the tissues were collected. No evidence of a persistent FVR virus infection was found in any of the tissues. Trigeminal ganglion explant cultures from six FVR virus carrier cats were shown to be latently infected with feline syncytia forming virus by treatment with 5-iododeoxyuridine. Five control cats had trigeminal ganglion explant cultures similarly treated but produced no evidence of infection. The possible significance of this is discussed.     

Temporary inhibition of neuronal apoptosis in Aujeszky's disease virus-infected swine

Previous studies have shown that during acute infection of the porcine trigeminal ganglia (TG), Aujeszky's disease virus (ADV)-infected neurons are protected from apoptosis induced by the virus itself and by cells of the immune system. However, TG neurons productively infected by ADV finally die and are phagocytosed by adjacent cells, a fact that leads us to speculate that the inhibition of neuronal apoptosis by ADV may be temporary rather than absolute. To address this issue we used TG and brain stem from pigs during acute infection by ADV. Infected cells were detected by immunohistochemical staining of viral antigens, whereas apoptotic cells were identified with an anti-active caspase-3 antibody, the TUNEL assay and by transmission electron microscopy. The results obtained in this study support the contention that the inhibition of neuronal apoptosis by ADV is temporary, since activation of caspase-3 could be detected in infected neurons at late stages in infection and because foci of advanced neuronophagia contained neurons exhibiting typical ultrastructural features of apoptosis.