In vitro markers to differentiate an avirulent from a virulent strain of aujeszky's disease virus

The susceptibility of the virulent NIA-2 and avirulent Bartha strains of Aujeszky's disease virus to various physical and chemical methods of inactivation was investigated. Both strains were highly sensitive to UV radiation and to various combinations of trypsin concentration and temperature. The Bartha strain was the more thermostable and pH-sensitive.     

Subclinical Aujeszky's disease virus infection in a pig herd and the characterisation of the strain of virus isolated

The spread of antibody to Aujeszky's disease virus through a susceptible pig herd was monitored after the probable introduction of infection by a recently purchased boar. The infection spread slowly through the herd but no clinical signs of Aujeszky's disease were seen. The strain of virus isolated was designated NIA-6. It has been characterised by a series of experimental infections and extends the known range of virulence of isolates of Aujeszky's disease virus made in Northern Ireland. The strain caused no disease in four-week-old piglets and is therefore less virulent than other isolates from Northern Ireland pigs. However, it killed rabbits and a proportion of experimentally infected two-week-old piglets, which differentiates it from the avirulent bovine isolate (NIA-4).     

Role of memory B-cell responses in serum and mucosal fluids of swine for protective immunity against pseudorabies virus.

We examined primary and memory isotype-specific antibody responses directed against pseudorabies virus in serum and mucosal fluids of pigs with and without passively acquired maternal antibody, and we studied the relationship between these responses and protection against virus challenge. Pigs were inoculated intranasally with the virulent NIA-3 strain or the avirulent Bartha strain, or they were inoculated IM with an inactivated vaccine containing the Phylaxia strain. Ten weeks later, all pigs were challenge-exposed intranasally with strain NIA-3. Only pigs that were without passively acquired antibody at the time they were inoculated with virulent virus appeared to have complete protective immunity against challenge exposure, as evidenced by lack of clinical signs of pseudorabies and lack of virus excretion. In contrast, pigs inoculated with strain Bartha or with the inactivated vaccine developed fever, had a period of growth arrest, and excreted virus after challenge exposure. In pigs without passively acquired antibody, intranasal inoculation with strains NIA-3 or Bartha was followed by primary IgM and IgA responses in serum and in oropharyngeal fluid as well as primary IgG1 and IgG2 responses in serum. Intramuscular inoculation with the inactivated vaccine induced primary serum IgM, IgG1, and IgG2 responses, but no mucosal responses. Challenge exposure of pigs that had been inoculated with the Bartha strain or the inactivated vaccine was followed by clear memory responses in serum and in oropharyngeal fluid. In contrast, challenge exposure of pigs that had been inoculated by the virulent NIA-3 strain was not followed by memory responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus

In the present study four attenuated virus strains, used as vaccines, and a virulent strain of Aujeszky's disease virus (ADV) were compared with respect to their virulence in mice, their ability to induce virus-specified thymidine kinase (TK) in infected cells, and their cleavage profiles of viral DNA's after treatment with the restriction endonuclease KpnI. The survival time of mice inoculated with the B-KAL or the virulent NIA-3 strain was comparable, whereas the Bartha and BUK strains required significantly longer periods to kill mice. Mice were resistant to the MK-25 strain of ADV. The strains were assayed for TK phenotype by plaque autoradiography after 3H-thymidine labelling of infected cells. MK-25 proved to be the only strain defective in induction of TK in pig kidney cells. Restriction endonuclease analysis of viral DNA's revealed that each vaccine strain showed a characteristic fragment pattern that could easily be differentiated from that of other vaccine and field strains of ADV. The present results demonstrate that the mouse virulence test and the TK assay detect differences in biological properties of ADV strains, but that restriction endonuclease analysis is required for unambiguous identification of vaccine and field strains of ADV.     

Pathological, ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (PEARS))

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6-day-old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re-isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum. Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

雏鹅新型病毒性肠炎病毒强毒在感染鸭胚体内形态发生学和宿主细胞超微病理学研究

将雏鹅新型病毒性肠炎病毒（NGVEV）强毒CH株经尿囊腔途径人工感染10日龄鸭胚,应用透射电镜和超薄切片技术研究病毒在宿主细胞内的形态发生及各组织器官的超微结构变化。结果表明：感染后不同时间剖杀及死亡鸭胚的尿囊膜、肠、心、肝、脑和肌胃组织中,均观察到60～70nm的病毒粒子。病毒粒子主要通过与细胞膜融合而进入细胞质内,然后在细胞核内进行复制和装配。最后病毒粒子通过核膜和细胞膜破裂的方式被释放。病毒侵害的主要靶细胞包括鸭胚尿囊膜上皮细胞、肠上皮细胞、肠道平滑肌细胞、成纤维细胞、肝细胞、肌胃黏膜上皮细胞和心肌细胞等,表现为细胞核内外膜间隙严重扩张,细胞质整体结构严重空化。病毒侵害的主要靶细胞器包括粗面内质网和线粒体,表现为粗面内质网扩张呈囊状;尿囊膜上皮细胞的线粒体出现固缩和异常聚集变化,而其他组织细胞的线粒体均表现为肿胀和嵴断裂、消失。本试验还发现NGVEV可诱导宿主细胞发生严重的细胞凋亡现象,表现为细胞皱缩,胞核内染色质密度增高,核固缩成一个或数个团块凝聚在核膜周边,胞质浓缩深染并形成凋亡小体。     

鸡传染性支气管炎病理形态学及发病机理的研究

１５０只１８日龄雏鸡随机分为两组，试验组９０只接种传染性支气管炎病毒（ＩＢＶ－Ｍ４１）后３５天内分３０批依次于不同时间扑杀，作组织病理学、超微结构及病毒抗原位检查，对照组６０只作相同的检查。结果表明，ＩＢＶ攻击的靶器官是气管，气管的病变表现为粘膜上皮细胞的损伤和脱、残留的在皮细胞增殖形成复层上皮、粘膜固有层及粘膜下层大量淋巴细胞浸润及粘膜逐渐恢复的相互连续的病理过程。肺脏初级、次级支气管也有类似的     

Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection

Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.     

Development of protein A-gold immunoelectron microscopy for detection of bovine coronavirus in calves: comparison with ELISA and direct immunofluorescence of nasal epithelial cells

A protein A-colloidal gold immunoelectron microscopy (PAG-IEM) technique was developed for the detection of bovine coronavirus (BCV) in the feces and nasal secretions of infected calves. Feces or nasal swab fluids were incubated sequentially with hyperimmune bovine anti-bovine coronavirus serum and protein A-gold, negatively stained, applied to formvar-coated copper grids and viewed using an electron microscope. The PAG-IEM method specifically identified BCV particles and possible subviral particles in feces and nasal-swab fluids from infected calves. The PAG-IEM method did not label other enveloped enteric viruses or morphologically similar fringed particles commonly found in feces. Detection of BCV using PAG-IEM was compared with ELISA and direct immunofluorescence (IF) of nasal epithelial cells by monitoring fecal and respiratory tract shedding of BCV from two experimentally infected and two naturally infected calves from birth to 3 weeks of age. PAG-IEM and ELISA detected shedding of BCV in fecal (4/4 animals) and nasal (3/4 animals) samples for an average of 5.25 days each. The observed agreement of BCV detection by PAG-IEM and ELISA was 85%. PAG-IEM may be a more sensitive immunoassay for the detection of BCV in diagnostic specimens from infected neonatal calves than ELISA. BCV infection of nasal epithelial cells was detected by immunofluorescence in 4/4 calves, persisted for the duration of the study in 2/4 calves and was sporadic in the other two animals. The observed agreement of BCV detection by PAG-IEM and IF was 57%.     

Pathological,ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: Porcine epidemic abortion and respiratory syndrome (PEARS))

Summary

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6‐day‐old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re‐isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum.

Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Tonsillar changes in pigs given pseudorabies (Aujeszky's disease) virus

All of the eight 5-day-old pigs orally given pseudorabies (Aujeszky's disease) virus developed tonsillitis. The initial changes occurred in the subepithelial area between the lymphoid nodule and the crypt epithelium, showing a characteristic pattern of necrosis. The necrosis became more severe and gained access into the lymphoid nodule and crypt epithelium. Coincident with the histopathologic changes, numerous specific immunofluorescences were detected, first in the nucleus and in some parts of the cytoplasm of cells distributed in the subepithelial area. The fluorescence subsequently spread into adjacent lymphoid nodules and crypt epithelial cells. Ultrastructurally, many enveloped virus particles were detected in the center of the necrosis. Thereafter, the crypt epithelial cells also underwent degeneration, and a small number of virus particles were detected in the nucleus of the degenerating epithelial cells. In the more advanced stage, the enveloped virus particles were discharged into the crypt lumen.     

Ultrastructural study of infectious bronchitis virus infection in infundibulum and magnum of commercial laying hens

The infundibulum and magnum of the oviduct were examined in hens in full lay which were infected with two Australian strains of infectious bronchitis virus (IBV). The ultramicroscopic changes in the infundibulum and magnum were compared with control hens which had eggs at different positions in the oviduct. The ciliated and granular cells of the surface epithelia and secretory epithelial cells of the tubular glands were the target cells of IBV. No pathological changes were recorded during 2-8 days post-infection (p.i.). Patchy loss of cilia occurred at 10-14 days p.i. Between 16 and 24 days p.i., there was no cilia loss and lymphoid nodules were observed in the muscularis layer of the infundibulum and magnum of some hens from both infected groups. Virus particles were detected mostly in the rough endoplasmic reticulum (RER) and Golgi complex between 10 and 12 days p.i. Cytopathology was noticed in various cell organelles between the 10th and 14th days p.i. There was an increase in RER deposits in infected cells, irrespective of egg position in the oviduct. The magnum was more affected than the infundibulum. Cellular changes were more severe in the infundibulum and magnum of T-infected hens as compared to N1/88-infected hens. Eggs with watery whites which were laid by infected hens could be attributed to cytopathological changes in the granular epithelial cells and tubular gland epithelial cells of the magnum resulting in reduced synthesis of albumen proteins. IBV can cause pathology in parts of the fully functional oviduct which may persist up to the 30th day p.i. However, both the challenge strains of IBV can cause a small number of hens to cease production. Loss of cilia in both the infundibulum and magnum pose a potential threat of secondary bacterial infection and also may affect fertility in breeder hens.     

人工感染IBDV鸡法氏囊的电镜研究

通过透射电镜系统观察了人工感染传染性法氏囊病病毒（ＩＢＤＶ）后鸡法氏囊各类细胞的病理变化。感染后１２￣２４ｈ，病毒粒子主要见于髓质淋巴细胞中，细胞中可见到大量纤维样病毒发生基质及无囊膜包围的大型病毒晶格，细胞核染色质浓缩，核中出现纤维样结构。感染后３６ｈ，淋巴细胞开始大量裂解死亡。无囊膜包围的病毒晶格也出现于髓质网状细胞中，被感染的网状细胞并不裂解，而表现出细胞凋亡的特征：染色质固缩呈颗粒块状，胞     

Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena)

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163⁺Sialoadhesin (Sn)⁺, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163⁺Sn⁺, CD163⁺Sn^- and to a lesser extent CD163^-Sn^- monocytic subtypes were involved in infection. CD163⁺Sn^- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163⁺Sn⁺ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163⁺Sn^- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena.     

Pathogenesis and Subsequent Cross‐Protection of Influenza Virus Infection in Pigs Sustained by an H1N2 Strain

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.     

2018年伪狂犬病病毒的流行特征及其遗传变异分析

为了了解当前伪狂犬病病毒（PRV）在我国猪群中的流行现状及其遗传变异情况,本研究利用PCR的方法对2018年1-12月来源于我国28个省、市、自治区的1 328份疑似猪伪狂犬病发病猪的组织样品开展了PRV-gE的检测及病毒的分离鉴定,同时针对所分离的病毒的gB、gC和gE基因进行PCR扩增和DNA测序,并展开遗传变异分析。结果显示,共有92份样品为PRV-gE基因检测阳性,阳性检出率为6.93%。从92份PRV-gE阳性样品分离到13株PRV。分别基于gB基因部分片段、gC和gE基因进行遗传变异分析发现13株PRV与我国2012年以后流行的毒株（如HeN1等变异株）亲缘关系较近,而与2012年以前所分离的毒株（如Ea等经典毒株）的亲缘关系较远;此外,绝大多数中国分离株与国外分离毒株（如NIA-3、Bartha、Kaplan等）在遗传进化树中位于不同的进化分支;相对于国外分离株及国内早期流行的经典毒株而言,13株PRV分离株的gB、gC和gE蛋白中均存在许多特征性的氨基酸位点变异。本研究对于了解我国PRV流行现状及当前流行的PRV毒株的生物学特征具有十分重要的意义。     

鹅副粘病毒病的研究

用ＳＰＦ鸡胚分别从扬州市不同患病鹅群的病鹅肝、脾等病料分离到５株病毒。鸡胚分离的病毒株均凝集鸡红细胞,ＨＡ为２７～１４,并被康复血清所抑制。毒价ＥＬＤ５０为１０１１．２４,人工感染易感鹅引起发病死亡,有与自然病例相同的症状和病变,并能在鹅群中流行传播。三株鸡胚绒尿液毒作负染电镜观察,病毒颗粒均呈大小不一,形态不正,表面有密集纤突结构,病毒内部由囊膜包裹着的螺旋对称的核衣壳,大小平均直径为１２０ｎｍ。根据血清学检测、病毒形态结构以及特性等初步的研究结果,本病分离毒株为副粘病毒科、副粘病毒属一员。用分离毒研制的灭活苗经试验表明有良好的保护率。     

Comparison of virus isolation, immunofluorescence and DNA probe hybridization for detection of pseudorabies virus in experimentally infected pigs.

Virus isolation, immunofluorescent staining and DNA probe hybridization, three techniques for the detection of pseudorabies virus (PRV) have been compared in pigs experimentally infected with the Thailand CB-1 strain PRV. The virus isolation and DNA hybridization detection demonstrated a good correlation in detecting infection in live animals by nasal swabbing. In white blood cells an earlier detection was seen with the DNA-hybridization techniques. Consistent results with all the three techniques tested were found in organ materials as nasal mucosa and tonsils as well as in the olfactory bulb.     

A scanning and transmission electron microscopic study of rotavirus-induced intestinal lesions in neonatal gnotobiotic dogs

Neonatal gnotobiotic dogs orally inoculated with canine rotavirus had ultrastructural changes limited to the jejunal and ileal regions of the small intestine. Early scanning electron microscopic findings consisted of swollen villus epithelial cells, denuded foci on intestinal villi, and slight to moderate villus atrophy. Later changes were slight villus atrophy with no denuded intestinal villi. Transmission electron microscopic changes in villus epithelial cells from 12 to 48 hours post-inoculation included: rotavirus particles associated with intracytoplasmic vacuoles near the terminal web and apical tubules; viral particles in dilated cisternae of rough endoplasmic reticulum; and moderate numbers of necrotic cells having no microvilli, swollen mitochondria, membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, and disruption of the cytoplasmic membrane. In jejunum and ileum at 72 to 154 hours post-inoculation, there were fewer necrotic villus epithelial cells and fewer virus particles. In addition, the ultrastructural morphology of the majority of the villus epithelial cells was similar to crypt epithelium. These studies showed that rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and destruction of villus epithelial cells.