Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs

OBJECTIVE: To evaluate the influences of animal age, bacterial coinfection, and porcine reproductive and respiratory syndrome virus (PRRSV) isolate pathogenicity on virus concentration in pigs. ANIMALS: Twenty-one 2-month-old pigs and eighteen 6-month-old pigs. PROCEDURE: Pigs were grouped according to age and infected with mildly virulent or virulent isolates of PRRSV. The role of concurrent bacterial infection was assessed by infecting selected pigs with Mycoplasma hyopneumoniae 21 days prior to inoculation with PRRSV. On alternating days, blood and swab specimens of nasal secretions and oropharyngeal secretions were collected. On day 21 after inoculation with PRRSV, selected tissues were harvested. Concentrations of PRRSV were determined by use of quantitative real-time PCR and expressed in units of TCID(50) per milliliter (sera and swab specimens) or TCID(50) per gram (tissue specimens). RESULTS: Concentrations of virus were higher in blood and tonsils of pigs infected with virulent PRRSV. Pigs infected with virulent PRRSV and M hyopneumoniae had significantly higher concentrations of viral RNA in lymphoid and tonsillar tissue. Coinfection with M hyopneumoniae resulted in a higher viral load in oropharyngeal swab specimens and blood samples, independent of virulence of the PRRSV isolate. Two-month-old pigs had significantly higher viral loads in lymph nodes, lungs, and tracheal swab specimens than did 6-month-old pigs, independent of virulence of the PRRSV isolate. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple factors affect PRRSV concentration in pigs, including pathogenicity of the PRRSV isolate, age, and concurrent infection with M hyopneumoniae.     

Comparison of the virulence of European and North American genotypes of porcine reproductive and respiratory syndrome virus in experimentally infected pigs

The objective of this study was to compare the virulence of Korean types 1 and 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolated from weaned pigs with respiratory disease. Affected pigs were within the same herd and animals infected with type 2 virus had significantly higher mean rectal temperatures than those with type 1 virus between days 2 and 9 post-inoculation (P < 0.05). Similarly, mean serum viral titres, expressed as tissue culture infective doses 50% (TCID₅₀)/mL, as well as macroscopic and microscopic pulmonary lesion scores, were significantly higher at multiple time points in pigs infected with type 2 PRRSV compared to those infected with type 1 virus. Mean numbers of PRRSV-positive cells/unit area of lungs and lymph nodes were also significantly higher in type 2 PRRSV infected pigs. This study demonstrates that type 2 PRRSV is more virulent than type 1 PRRSV in this experimental setting as reflected by the pulmonary pathology induced, the extent of virus distribution, and oral shedding of the virus.     

Experimental quantification of the transmission of Porcine reproductive and respiratory syndrome virus

We conducted an experiment to determine the ability of vaccine against Porcine reproductive and respiratory syndrome virus (PRRSV) to reduce the transmission of PRRSV among pigs. At the end of the experiment, transmission rates did not differ significantly (P = 0.61) between the vaccinated and nonvaccinated pigs, the mean R-values being 0.598 (95% confidence interval [CI] 0.136 to 3.218) and 0.264 (95% CI 0.008 to 2.266), respectively. The unusually low rate of PRRSV transmission in both groups may not have provided a sufficient challenge to detect a vaccine effect. Several factors could affect the rate of PRRSV transmission: isolate virulence, inoculation dose, inoculation route, number of passages of the challenge virus in cell culture, and population size. Of these, isolate virulence appears to be the most important factor associated with the low transmissibility observed in this study. More studies comparing rates of transmission between PRRSV isolates with diverse levels of virulence are needed to better understand this association.     

Effects of challenge with a virulent genotype II strain of porcine reproductive and respiratory syndrome virus on piglets vaccinated with an attenuated genotype I strain vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in most parts of Asia, where genotype I and II strains of diverse virulence may coexist. This study evaluated the outcome of infection with a highly virulent Asian genotype II PRRSV isolate in piglets vaccinated with a genotype I vaccine. Twenty-one 3-week-old piglets were divided in three groups: Pigs in group V (n=8) were vaccinated with an attenuated genotype I commercial PRRSV vaccine, while pigs in group U (n=8) and a control group (group C; n=5) were unvaccinated; 6 weeks later, pigs in groups V and U were challenged intranasally with a highly virulent strain of genotype II PRRSV (1×10(5) 50% tissue culture infectious doses/mL), while pigs in group C received a placebo. Over a period of 21 days after challenge, vaccinated pigs had significantly lower mortality (0/8 versus 2/8), fewer days of fever, a lower frequency of catarrhal bronchopneumonia, higher weight gains (13.4 versus 6.6 kg) and lower levels of viraemia compared to unvaccinated challenged pigs. Immunisation with a genotype I attenuated PRRSV vaccine provided partial protection against challenge with a highly virulent genotype II strain.     

Comparative pathogenicity of type 1 and type 2 isolates of porcine reproductive and respiratory syndrome virus (PRRSV) in a young pig infection model

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates are classified in two different genotypes, based on genomic heterogeneity: type 1, which comprises European type isolates, and type 2, which includes North American type isolates. It is believed that members of both genotypes differ in some biological properties including pathogenicity, however extensive studies comparing isolates of both genotypes have never been carried out. The objective of the present study was to compare the pathogenic properties of six different PRRSV isolates, three of type 1 and three of type 2, in a young pig infection model. For this purpose, a total of 105 3-week-old piglets were divided in 7 groups of 15 animals that were exposed on day 0 of the experiment to one of the six isolates tested or were mock infected (negative control group). Clinical signs and rectal temperatures were recorded daily and blood samples were taken on days 3, 6, 9, 12, 15, 18 and 21 of the experiment. On days 7, 14 and 21 post-inoculation five animals per group were sacrificed, macroscopic lung lesions were evaluated and different tissue samples were collected to determine viral organic distribution. The results obtained indicate that type 2 isolates are more pneumovirulent than type 1 isolates, as demonstrated by the recording of respiratory clinical signs only in pigs exposed to type 2 viruses and by the severity of macroscopic and microscopic lung lesions in those pigs. However, no clear differences could be established between genotypes in systemic clinical signs or viral load and viral distribution after challenge. These results support the general idea that type 2 isolates induce more severe respiratory disease than type 1 isolates.     

猪繁殖与呼吸综合征病毒ORF5基因的遗传变异和系统进化分析

猪繁殖与呼吸综合征病毒(PRRSV)毒株间变异广泛,而且这种变异能够筛选出强毒株。在结构蛋白中GP5的变异率最高,其变异能够导致病毒毒力的变化。为了确定PRRSV在我国的进化趋势和毒力增强的原因,对从2003年以来分离到14株PRRSV GP5蛋白的核苷酸序列和推导的氨基酸序列进行分析并构建了系统进化树。结果显示2006年以来从国内分离的毒株其糖基化位点发生了明显改变,并且在系统进化树上形成了新的分支,进化分析显示它们可能来源于1995年的中国分离株CH-1 a。     

Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus

OBJECTIVE: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs. SAMPLE POPULATION: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV. PROCEDURE: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined. RESULTS: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs. CONCLUSIONS AND CLINICAL RELEVANCE: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.     

Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n=8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n=9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n=9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n=9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n=9), pigs infected with VR-2385 (n=9), and pigs infected with NC16845b (n=9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.     

Proinflammatory cytokines and viral respiratory disease in pigs

Swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) are enzootic viruses causing pulmonary infections in pigs. The first part of this review concentrates on known clinical and pathogenetic features of these infections. SIV is a primary respiratory pathogen; PRCV and PRRSV, on the contrary, tend to cause subclinical infections if uncomplicated but they appear to be important contributors to multifactorial respiratory diseases. The exact mechanisms whereby these viruses cause symptoms and pathology, however, remain unresolved. Classical studies of pathogenesis have revealed different lung cell tropisms and replication kinetics for each of these viruses and they suggest the involvement of different lung inflammatory responses or mediators. The proinflammatory cytokines interferon-alpha (IFN-alpha), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) have been shown to play key roles in several respiratory disease conditions. The biological effects of these cytokines and their involvement in human viral respiratory disease are discussed in the second part of this review. The third part summarises studies that were recently undertaken in the authors' laboratory to investigate the relationship between respiratory disease in pigs and bioactive lung lavage levels of IFN-alpha, TNF-alpha and IL-1 during single and combined infections with the above viruses. In single SIV infections, typical signs of swine "flu" were tightly correlated with an excessive and coordinate production of the 3 cytokines examined. PRCV or PRRSV infections, in contrast, were subclinical and did not induce production of all 3 cytokines. Combined infections with these 2 subclinical respiratory viruses failed to potentiate disease or cytokine production. After combined inoculation with PRCV followed by bacterial lipopolysaccharide, both clinical respiratory disease and TNF-alpha/IL-1 production were markedly more severe than those associated with the respective single inoculations. Taken together, these data are the first to demonstrate that proinflammatory cytokines can be important mediators of viral respiratory diseases in pigs.     

Effects of different us isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on blood and bone marrow parameters of experimentally infected pigs

Seventy five-week-old, crossbred, caesarean-derived, colostrum-deprived pigs were randomly divided into five groups of 14 pigs and assigned one of five treatments: the intranasal inoculation of 1 (5.7) TCID50 of one of four plaque-purified isolates of porcine reproductive and respiratory syndrome virus (PRRSV) (VR2385, VR2431, ISU-984 and ISU-22), or uninfected cell culture and media. Haematological variables were measured for 21 days and bone marrow was analysed when the pigs were killed three, seven, 10, 21 or 28 days after the inoculation. The PRRSV-infected pigs had non-regenerative anaemia and markedly increased myeloid:erythroid ratios from three to 21 days after inoculation. There was a significant (P < 0.05) difference in the severity of the anaemia induced by the four PRRSV isolates; the most highly pneumovirulent strains (VR2385, ISU-984 and ISU-22) induced more severe anaemia than the least virulent isolate (VR2431). The anaemia induced by PRRSV was probably due to a direct or indirect effect on erythroid precursor cells in the bone marrow.     

Pathogenic Analysis of Two Strains of Porcine Reproductive and Respiratory Syndrome Virus

Samples of sick pigs suspected of porcine reproductive and respiratory syndrome (PRRS) infection were collected from Hebei and Fujian provinces in China in 2016. Studies were carried out to investigate the variation characteristics of the isolated virus strains.The virus isolates were propagated in Marc-145 cells and caused syncytial cytopathic effect. RT-PCR and indirect fluorescent assay (IFA) results showed that the isolates were HP-PRRSV and hence designated as PRRSV CZ16A and NP16A, respectively. In order to study the pathogenicity of CZ16A and NP16A, the isolated strains were purified by plaque-isolation, and then inoculated to 60-days-old healthy pigs, which were negative in both of PRRSV infection and antibody. Clinical observation, histopathological changes and viremia detection were carried out to evaluate the pathogenicity of these two strains of PRRSV. Results showed that both isolates could replicate in pigs and cause a high body temperature, but these two strains of PRRSV isolates could not cause morbidity or mortality in experimental animals.Autopsy results also showed that NP16A could cause consolidation of the lung, lymph node enlargement in tested animals, suggesting that its virulence was a little stronger than CZ16A.     

2株猪繁殖与呼吸综合征病毒的致病性分析

为追踪猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）分离株的变异特点,本研究于2016年从河北和福建疑似猪繁殖与呼吸障碍综合征（PRRS）发病猪场采集病料,接种Marc-145细胞,产生合胞体样细胞病变,RT-PCR和间接免疫荧光试验鉴定为HP-PRRSV,将其分别命名为PRRSV CZ16A和NP16A。为研究PRRSV分离株CZ16A和NP16A对猪的致病性,将分离毒株经噬斑纯化后分别接种PRRSV抗原、抗体双阴性的60日龄健康仔猪,通过临床观察、解剖病理变化及病毒血症检测等指标初步探讨两株PRRSV的致病性。结果表明,分离株CZ16A和NP16A感染猪后均能够在动物体内复制,并引起体温升高,但两株PRRSV分离株均未引起试验动物的发病和死亡,剖检结果显示NP16A分离株能引起动物肺部实变、淋巴结肿大,其毒力比CZ16A分离株稍强。     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.     

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.     

Identification and clinical assessment of suspected vaccine-related field strains of porcine reproductive and respiratory syndrome virus

OBJECTIVE: To determine the origin and clinical relevance of selected strains of porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV). ANIMALS: 38 pigs without antibodies for PRRSV. PROCEDURE: A seemingly uncommon restriction endonuclease digestion site in a commercially available vaccine strain of attenuated PRRSV was tested for its stability and prevalence under defined conditions. Selected field strains of PRRSV, with or without the restriction-site marker, were subsequently tested in pigs for virulence and for their ability to replicate competitively in pigs simultaneously given the vaccine. RESULTS: Under experimental conditions, the restriction-site marker was stable during long-term infection of pigs. It was not detected in any of the 25 field strains of PRRSV that were isolated before use of the vaccine or 21 of 25 field strains that were isolated after use of the vaccine but that, on the basis of previous testing, were believed unrelated to the vaccine strain. Conversely, it was detected in 24 of 25 field strains that were isolated after use of the vaccine and that, on the basis of previous testing, were believed to be direct-line descendants of the vaccine strain. Putative vaccine-related strains caused more pronounced pathologic changes than did the vaccine strain alone, and they predominated during replication in pigs also given the vaccine strain. CONCLUSIONS: In some swine herds, the vaccine strain may have persisted and mutated to a less attenuated form. CLINICAL RELEVANCE: The potential for persistence and mutation of specific strains of virus should be an important consideration when designing vaccination programs involving attenuated PRRSV.     

Effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs

OBJECTIVE: To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. ANIMALS: Four hundred forty 6- to 8-week-old PRRSV-na?ve pigs. PROCEDURES: Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). RESULTS: Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.     

Efficacy of vaccination with porcine reproductive and respiratory syndrome virus following challenges with field isolates in Japan

The objective of this study was to evaluate the influences of genetic and antigenic variations in field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on vaccine efficacy. Four-week-old pigs were vaccinated with a commercial modified live virus vaccine. Four weeks after vaccination, pigs in both the vaccinated group and the non-vaccinated group were challenged intranasally with 10(7) TCID(50) of PRRSV wt-11 (Experiment 1) or PRRSV wt-7 (Experiment 2). Based on genome sequencing of ORF5 and cross neutralization test results, PRRSV wt-11 is similar to the vaccine strain, whereas wt-7 is distinct from the vaccine strain. In the vaccinated challenged groups, clinical signs were less severe, the mean rate of weight gain was greater, and gross lung lesions were less severe when compared with the non-vaccinated challenged groups in both experiments. In Experiment 1, the virus was isolated from serum at 3 days post-challenge, and the mean virus titers in broncho-alveolar lavage fluids (BALF) and tissues were lower in pigs in the vaccinated challenged groups compared with those in the non-vaccinated challenged group. In Experiment 2, virus isolation from serum, BALF and tissues showed no significant differences between the groups. These results suggest that commercial PRRSV vaccine could be effective in reducing clinical disease following a challenge with field isolates of PRRSV. However, with regards to virological protection, the efficacy of the vaccine may be affected by the nature of the PRRSV isolates.     

Analysis of glycoprotein I (gI) negative and aberrant pseudorabies viral diagnostic isolates.

Glycoprotein I (gI) phenotypes and genotypes of 4 pseudorabies viral diagnostic isolates were evaluated by use of in vitro DNA amplification, monoclonal antibody binding, gI-specific serodiagnostic responses, and in vivo virulence approaches. Three viruses were avirulent and did not elicit gI-specific serologic responses, react with gI-specific monoclonal antibodies, or contain gI epitope-encoding DNA sequences. The fourth virus was virulent and did elicit a gI-specific serodiagnostic response. Compared with reference virulent pseudorabies viruses, however, the fourth isolate had reduced reactivity with a group of gI monoclonal antibodies and had a single nucleotide sequence substitution with a corresponding putative amino acid change in the epitopically dominant portion of the gI molecule. Presumably, the first 3 isolates represented diagnostic recoveries of viruses derived from gI-deleted modified-live pseudorabies viral vaccines, whereas the fourth isolate was a virulent but gI-aberrant wild-type virus. Thoroughly assessing the gI status of pseudorabies viral diagnostic isolates was considered to be essential in evaluating the epidemiologic importance of these viruses and in monitoring the validity of gI-based vaccine companion tests now used worldwide in pseudorabies control and eradication programs.