In the present study, experiments were conducted to assess the potential role of the EUs4 gene product gp2 in the severe respiratory inflammatory response observed following intranasal infection of CBA mice with pathogenic EHV-1. We report here that the expression of the full-length RacL11 gp2 (791 amino acids) by a recombinant KyA virus completely restores the ability of this otherwise-fully attenuated KyA strain to elicit fatal immunopathological responses in the lower respiratory tract, indicating a vital role of gp2 in EHV-1 pathogenesis.
Our hypothesis that gp2 may play an important role in EHV-1 pathogenesis came from studies using a recombinant RacL11 (RacL11ΔgIgE) with glycoproteins gI and gE deleted and a recombinant KyA (KgIgE), in which gI and gE were restored to the attenuated KyA background (15
). In that study, the expression of gI and gE by KyA did not restore full virulence as KgIgE-infected mice exhibited only a moderate weight loss and, in contrast to RacL11-infected mice, did not succumb to infection by day 6 postinfection. The lack of severe respiratory illness and survival of these mice, however, finally resulted in spread of KgIgE to the brain, an outcome directly attributed to the expression of gI and gE (14
). Surprisingly, the deletion of gI and gE from RacL11 (RacL11ΔgIΔgE) resulted in a course of respiratory illness virtually indistinguishable from that following infection with the pathogenic RacL11 strain (15
). Taken together, these results suggested that RacL11 expresses a virulence factor(s) that is not present in KyA and is capable of eliciting severe respiratory disease in the absence of gI and gE expression.
Over years in cell culture, KyA has undergone several genomic deletions, the most prominent in the unique-short genomic region being deletions of EUs6
(gE), and more than half of the EUs4
open reading frame (8
). The failure of KgIgE (lacking full-length gp2) to elicit severe respiratory illness and the ability of RacL11ΔgIΔgE that expresses gp2 of 791 amino acids to elicit severe respiratory illness led to the hypothesis that the expression of full-length gp2 was a critical factor in the induction of severe respiratory immunopathology. The results presented here strongly support this hypothesis.
The loss of 20% total body weight and the severe clinical signs observed in KyARgp2F-infected mice are similar to those following infection with the highly pathogenic RacL11 (this study and references 9
). The massive influx of inflammatory cells into KyARgp2F-infected lungs, which was preceded by increased levels of mRNA specific for MIP-1α, MIP-1β, MIP-2, and TNF-α, mimics the steps of pathogenesis observed in RacL11 infection (32
). The observation that greater than 30 transcripts encoding factors that function in innate immunity and inflammation were increased in lungs of mice infected with KyARgp2F versus KyA (Fig. ; Tables and ) precedent to the influx of large numbers of macrophages strongly suggests a role for gp2 in this immunopathology. That lower levels of transcripts encoding chi313, an inhibitor of IL-1 and TNF-α (23
) and a potent recruiter of eosinophils (29
), were present in KyARgp2F-infected lungs is consistent with the absence of eosinophilia following infection with either RacL11 (32
) or KyARgp2F (data not shown). In the context of the release of potent proinflammatory and inflammatory factors, it is noteworthy that gp2 has been shown to be cleaved into two subunits, the 42-kDa C-terminal membrane-bound moiety and a larger secreted form. It is conceivable that soluble gp2 may function as a signaling molecule, and this function is currently being addressed by generating mutant viruses that are unable to express the secreted form of the protein.
Previous reports assessing the role of gp2 in EHV-1 virulence yielded conflicting results. In one study the deletion of EUs4
from the pathogenic Ab4 strain reduced virulence, as infected mice exhibited no clinical signs and lost only moderate amounts of body weight (24
), suggesting a role for gp2 in respiratory virulence. The finding of a complete reduction of virulence was corroborated in another study using gp2-negative RacL11, which was completely apathogenic for BALB/c mice. However, in the same study, infection of mice with KyARgp2F expressing the full-length RacL11 gp2 failed to result in a significant weight loss (38
), raising questions regarding the role of gp2 in EHV-1 pathogenesis. There are three significant differences in the previous studies and the work presented here. First, the dosage applied to mice here was fourfold higher. Second, in contrast to the use of BALB/c mice in the two studies mentioned above, this investigation employed CBA mice which were found to be more susceptible to EHV-1-mediated disease than either the C57BL/6 or BALB/c mouse strains (references 14
, and 33
and personal observation). To directly address this question, 3- to 4-week-old BALB/c mice were weighed immediately prior to infection with the higher dose of 4 × 105
PFU of KyA, KyARgp2F, or RacL11, weighed daily thereafter, and monitored for clinical signs of respiratory disease. In a pattern of weight loss almost identical to that observed in CBA mice, KyARgp2F- and RacL11-infected BALB/c mice exhibited a mean total body weight loss of 21 and 22%, respectively, on day 4 postinfection, when the experiment was terminated (data not shown). BALB/c mice survived 1 day longer than CBA mice infected with the same dose, indicating a possible influence in the strain of mouse used. These results strongly suggested that the higher dose of 4 × 105
PFU/mouse in this study versus 105
PFU/mouse in the previous study (38
) may be the critical factor responsible in the discrepancies in outcomes described in these two studies.
Important questions remain to be answered regarding the role of gp2 in EHV-1 virulence. First, it is not known whether cleavage of full-size gp2 to a highly O-glycosylated N-terminal region and a 42-kDa C-terminal subunit (39
) is required for virulence. Second, the specific region(s) of full-size gp2 essential for virulence has yet to be identified. A recent study comparing gp2 of various strains and isolates of EHV-1 and EHV-4 noted extensive polymorphism (17
) in the reiterated repeat region toward the N terminus (Fig. ). Our findings that gp2 of two pathogenic strains, RacL11 and its counterpart in Ab4 (36
), both harbor this polymorphic region and that gp2 of attenuated KyA lacks the entire variable region are consistent with the hypothesis that this polymorphic region may play a role in determining EHV-1 biological properties.
A recent study reported that inoculation of BALB/c mice with DNA encoding gp2 resulted in a slight reduction in viral lung titers upon subsequent virus challenge and suggested the inclusion of gp2 in a subunit vaccine and its importance as a component of whole-virus vaccines (22
). The results in the present study indicate caution regarding the inclusion of full-length gp2 in EHV-1 vaccines until a better understanding is obtained concerning the role of this large glycoprotein in infection and the mechanism(s) by which it elicits inflammation in the respiratory tract.