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Experimental quantification of transmission of genetically engineered pseudorabies virus
Vaccine, Vol. 13, No. 18, pp. 1763-1769,
1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264-410X/95 $iO+O.OO
0264-410X(95)00156-5
Experimental quantification of transmission of genetically engineered pseudorabies virus W.A.M. Mulder*Q, M.C.M. De Jong*, J. Priem*, J.M.A. Pal* and T.G. Kimman*
A. Bouma*,
There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent d@erent PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PR V mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PR V vector vaccine (gE- , TK- , gG- ) that expresses the glycoprotein El (El) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when RI, the infection can spread; when R
virus mutant
strains;
experimental
transmission;
Pseudorabies virus (PRV) (synonyms: Aujeszky’s disease virus, suid herpesvirus-1) is a highly neurotropic alphaherpesvirus that causes Aujeszky’s disease in pigs’-‘. Swine are the natural host for this virus, its sole reservoir, and the sole source of virus transmission. PRV is prevalent in most parts of the world and causes economic losses in the swine industry and leads to trade barriers between countries. In the Netherlands and other countries of the European Community, PRV eradication programmes have started using marker vaccines that enable serologic differentiation between vaccinated and *Institute for Animal Science and Health, Department of Porcine and Exotic Viral Diseases and Department of Pathobiology and Epidemiology, P.O. Box 365, 8200 AJ Lelystad, The Netherlands. tuniversity of Utrecht, Department of Veterinary Pathology, Yalelaan 1, 3508 TD Utrecht, The Netherlands. $To whom all correspondence should be addressed. (Received IO February 1995; revised 30 June 1995; accepted 14 July 1995)
pigs
infected pigs4,‘. The current PRV vaccines protect against severe clinical signs of disease but usually do not prevent virus multiplication and excretion or the establishment of latency after infection6”. Vaccines against PRV should not only be applied to prevent the clinical consequences of a field infection, but first and foremost these vaccines should be applied to reduce the transmission of PRV among pigs, thus supporting the eradication of the virus’. In most eradication programmes only marker vaccines that lack expression of glycoprotein E (gE) are allowed. In addition, some vaccines lack expression of thymidine kinase (TK) to further decrease the virulence of the vaccine. Glycoprotein E promotes cellto-cell spread of the virus in vitro”“. In vivo gE is important for virulence and involved in invasion of the central nervous system”p’3. The viral TK functions in the salvage pathway for the supply of deoxynucleotides and is also important for expression of virulence14,‘5. Besides for eradication of PRV, PRV vaccines can be used as vector vaccines that express one or more
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1995 Volume
13 Number
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Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
Table 1
Transmission
Experiment/PRV 1. 2. 3.
M205 M205 Ml41 M206 Ml41 M207
of intranasally
strain
(gE-, TK-, gG-, El’) (gE-, TK-, gG-, El+) (aE-) (GE-; TK-, gG-) (gE-) (TK-)
inoculated
PRV mutant strains among pigs Average duration of excretion= (days)
Average daily t&e6 (log,,, p.f.u. per g OPF)
Number of pigs with neutralizing Ab
Number of virus-excreting
in.
con.
in.
con.
in.
con.
in.
con.
5’ 5 5 5 5 5
1 0 5 0 5 5
5 :
0 0 3 0 5 5
3.2 4.0 10.4 4.4 10.2 9.8
?I 12.7 0 12.2 11.8
4.7 4.9 5.6 4.5 6.1 6.4
: 6.0 0 5.6 6.5
Z 5
pigs
aThe number of days from the first until the last day of PRV excretion averaged over those pigs that did excrete PRV on at least one day. bArithmetic average titre over the days on which the pig did excrete PRV. ‘Each of figures in this and the next three columns refer to a total of 5 pigs. in=innocukted pigs and co&contact-exposed pigs
heterologous genes encoding immunorelevant proteins of other microorganisms’6.’ . Recently, the application of PRV as an efficient vector vaccine was demonstrated by Van Zijl et al.“, who showed that immunization with PRV recombinants that expressed the envelope El (El) of hog cholera virus (HCV) protected pigs against both pseudorabies and classical swine fever. Before these live vector vaccines can be applied, potential harmful sideeffects need to be evaluated”. Previously we reported that the expression of El by PRV did not change cell or host tropism, nor did it change the virulence of either non-virulent or virulent PRV strains2’,“. In this study we used a small-scale experimental model’ to estimate the transmission among susceptible pigs of PRV mutant strains that lack gE or TK, and of the PRV vector vaccines (gE , TK ~, gG-) with or without El of HCV. The estimated number of secondary cases per infectious individual, i.e. the reproduction ratio R, was used to estimate the spread from inoculated animals to susceptible contact-exposed animals. The ratio R has a threshold property; when Rl, the infection can spread; when R< 1, the infection will disappear22~24. We subsequently investigated whether gE-negative PRV could also be transmitted in a population which is co-infected with wild-type PRV. Therefore, experiments were performed to determine whether co-inoculation of a gE-negative and a gE-positive PRV strain affected the individual transmission of these strains.
MATERIALS
AND METHODS
Virus strains
The virulent wild-type strain NIA-325 was used as a parent strain for the development of the mutant viruses. Mutant viruses were generated by overlap recombination of four to five DNA fragments together compromising the entire PRV genome and insertion mutagenesis using an oligonucleotide with translational stop codons in all reading frames and an EcoRI site as described26.27. Mutant virus strains were plaque-purified three times on SK-6 cells2s and their integrity was confirmed by restriction enzyme analysis of the linker insertion sites. The PRV sequences immediately flanking the oligonucleotide insertion sites did not contain alterations. The gE-negative PRV strain (M141) has a deletion of 1729 base pairs (bp) in the gene encoding gE. The deletion was generated between the DraI site in the 5’ end of the gE gene and the EcoRI site of an oligonucleo-
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Vaccine 1995 Volume 13 Number 18
tide insertion mutant in the 3’ end of the gene, deleting nucleotides 6833-856226. The TK-negative strain (M207) contains a deletion of 19 bp in the 3’ part of the UL23 gene encoding TK. The 19 bp deletion is identical to the deletion in the vaccine strain 78329. The strain lacks functional TK activity and is completely nonvirulent for mice. The construction and characterization of strain M205 (gE-, TK-, gG_, El’), the vector vaccine expressing El of HCV, and of the control strain M206 (gE _, TK _, gG - ) without El, has been described previously’*. The gene encoding El of HCV was inserted into the glycoprotein gG (gG) locus. Expression of glycoprotein El of HCV did not change the virulence or pathogenesis of this PRV vector virus in vivo20.
The gE-positive PRV strain (Sterksel), a mildly virulent field isolate”, was used for the co-inoculation experiment with the gE-negative strain.
Animal experiments
Dutch Landrace pigs were obtained from the specificpathogen-free (SPF) herd of the Institute for Animal Health and Science. The pigs were born to unvaccinated sows and had no antibodies against PRV at the start of the experiment. Four experiments were performed independently; the first three experiments were designed to estimate the transmission of PRV mutant strains (Table 1) and the fourth experiment was designed to investigate a possible competition in transmission between gEnegative and gE-positive PRV strains (Table 2). The transmission of the vector vaccine M205 (gE -, TK- , gG-, El’) expressing El was investigated once after intramuscular inoculation, because this route is mostly used for vaccination against PRV. In all other experiments, PRV strains were intranasally inoculated, because this represents the natural route of infection of PRV and represents the “worst-case” route by which the virus is transmitted. To investigate a possible competition in transmission (exp. 4) a gE-negative strain (M141) and a gE-positive strain (Sterksel) were coinoculated in pigs. The individual transmission of these co-inoculated strains was compared with the transmission of separately inoculated gE-negative or gE-positive PRV strains. Groups of ten 3-week-old pigs were placed in separate isolation units at a density of 0.85 m2 pig-‘. Five pigs, randomly chosen from the ten pigs in each group, were
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al. Table 2
Competition
in transmission
between
intranasally
Number of virus-excreting
pigs
co-inoculated
gE-negative
and gE-positive
PRV strains
Average duration of excretiona (days)
Average daily titreb (log,, p.f.u./g OPF)
in.
con.
in.
con.
in.
con.
Co-inoculated: Ml41 (gE-) Sterksel (gE+)
5= 5
3 5
9.2 13.6
5.3 7.0
4.6 5.5
4.3 5.8
Separately inoculated: Ml41 (gE-) Sterksel (gE+)
3 5
z
11.3 14.0
3.7 7.6
6.1 5.6
6.5 5.4
Inoculation/PRV
strain
7he number of days from the first until the last day of PRV excretion averaged over those pigs that did excrete PRV on at least one day. *Arithmetic average titre over the days on which the pig did excrete PRV. “Each of figures in this and the next three columns refer to a total of 5 pigs. in=innoc&ted pigs and co&ontact-exposeb pigs
placed in a separate cubicle. The remaining 5 pigs of each group were inoculated with 10’ plaque forming units (p.f.u.) virus in the first three experiments (Table I), or with lo5 p.f.u. virus in the competition experiments (Table 2). Virus suspension (0.5 ml) was slowly administered into each nostril during inspiration. After 24 h, pigs were reunited to their previous companions, thereby contact-exposing these other 5 pigs to a PRV mutant strain excreted by the inoculated pigs. Thus, each group consisted of 5 inoculated (possible infectious) pigs and 5 contact-exposed (susceptible) pigs. The experimental conditions (e.g. density, origin of pigs, sampling) were similar in all the experiments. Sampling procedures
Starting the day before inoculation, and then each day for 16 days after inoculation, swabs of oropharyngeal fluid (OPF) were collected from all animals to measure virus excretion. For detection of neutralizing antibodies against PRV, serum blood was taken once before inoculation, and then weekly until the end of the experiment, 5 weeks after inoculation. Swabs were used to collect OPF samples. Swabs were extracted with 4 ml Dulbecco’s modification of Eagle’s medium supplemented with antibiotics and 2% fetal calf serum as described previously, and were stored at - 70”C2’. Subsequently, OPF samples were titrated for virus by plaque assay on monolayers of SK-6 cells. In experiment 4 an immunoperoxidase monolayer assay was performed on infected monolayers using MAb 2 that detects a conformational dependent epitope on the antigen binding domain E of gE”. Therefore, in OPF samples of the co-inoculated group, we could discriminate plaques caused by gEpositive virus (Sterksel strain) and plaques caused by gE-negative virus (strain M141). Virus titres are expressed as log,, p.f.u. per g OPF. Serum samples were stored at - 70°C and were tested for virus neutralizing antibodies against PRV as described by De Leeuw et ~1.“~Detection of infectious PRV in OPF samples or induction of neutralizing antibodies against PRV was considered evidence of infection. Statistical
methods
To estimate transmission of virus within experimental groups of pigs, we used the stochastic SusceptibleInfectious-Removed (SIR) model, described elsewhere’. The extent of transmission is expressed as the reproduction ratio R, which is mathematically defined as the
average number of new infections caused by one infectious individual during its entire infectious period”. Given the outcome of the observed outbreak in a population, the reproduction ratio R can be estimated, using a martingale estimatorX4:
The variables are the total number of pigs in the population (N), the number of susceptible pigs at the start of the infection chain (So), the number of susceptible pigs at the end of the infection chain (D), and the percentage of infectivity that is left, when the last susceptible pig becomes infected (Z). When all susceptible pigs become infected (S,=O), 2 is used to make a correction, because a part of the infectivity may not have been used after the last contact-infection. 2 was estimated by the amount of excreted virus from the moment of the last contact-infection until the end of the experiment divided by the total amount of excreted virus. The threshold value R predicts whether or not the infection will spread. When Rl, the infection can spread; when R< 1, the infection will disappear22p24. The algorithm for calculating exact probabilities of the final outcomes has been described previously. The exact probability distribution for R=l was used to test whether the R was larger than one, and the distribution based on the pooled estimate was used to compare transmission between virus strains’. For multiple paired comparisons of R within experiments, we did not use P=O.O5 as a criterion for significance but P=O.Ol. Differences in duration of virus excretion and virus titres in the OPF were tested with the Mann-Whitney U test. RESULTS Transmission
of PRV mutant strains
The results of the experiments designed to estimate the transmission of PRV mutant strains that lack gE or TK, or that express El of HCV are summarized in Table I. Pigs were inoculated intranasally, because this represents the natural route of infection of PRV and represents the “worst-case” route by which the virus can be transmitted. In the experiments, induction of
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Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
neutralizing antibodies against PRV and virus excretion were measured, and both were used for the detection of infection. As can be seen from the results in Table I, detection of virus excretion appears less sensitive than detection of neutralizing antibodies. In some cases, pigs developed neutralizing antibodies, but virus excretion was not detected. This occurred for 1 pig contactexposed to the vector vaccine M20.5 (gE ~ , TK ~ , gG _ , El+) expressing El in experiment 1, and for 2 pigs contact-exposed to the gE-negative virus in experiment 2. Both the inoculated and contact-exposed pigs excreted the gE-negative PRV strain (M141) for a long period (lo-12 days, Table I), except the contact-exposed pigs in experiment 4 that excreted the virus for a shorter period (3.7 days, Table 2). The pooled estimate for the reproduction ratio for the three independent experiments of the gE-negative PRV strain was 10.1 (S.E. 4.98), and was significantly 1 (P=O.O005). Furthermore, in experiment 2, the reproduction ratio of this gEnegative strain was significantly larger than the R for vector vaccine strain M205 (gE_, TK-, gG_, El+) (P=O.O075). In experiment 3, the R of the gE-negative strain was significantly larger than the R of the vaccine strain M206 (gE_ , TK-, gG-) (P=O.O006); R of the gE-negative strain was not significantly larger than the R of the TK-negative PRV strain (M207) (P=O.ll). Thus, these results indicate that a gE-negative PRV strain can spread in a susceptible, i.e. sero-negative, pig population. In experiment 3, both inoculated and contact-exposed pigs excreted the TK-negative PRV strain (M207) for a similar long period as the gE-negative strain. The estimated R for the TK-negative strain was 5.0 (S.E.=3.53). In this experiment, the reproduction ratio for the TKnegative PRV strain was not significantly 1 (P=O.O813). The R of the TK-negative strain was not significantly larger than the R of the vaccine strain M206 (gE -, TK - , gG ~ ) (P=O.O374). In experiment 1, contact-exposed pigs were not infected after intramuscular inoculation of pigs with the PRV vector vaccine M205 (gE _, TK _, gG _, El+) (data not shown). The intramuscular inoculated pigs developed neutralizing antibodies, but did not excrete the virus (data not shown). So, the estimated R for the vector vaccine M205 (gE ~, TK ~, gG _, El+) after intramuscular inoculation was 0. In contrast to the intramuscular route, intranasally inoculated pigs excreted the vector vaccine in both experiments 1 and 2. The period of virus excretion was short; on average, respectively, 3.2 and 4.0 days. None of the contactexposed pigs excreted virus, but 1 contact-exposed pig in experiment 1 developed neutralizing antibodies. The pooled estimate for the reproduction ratio for the intranasally inoculated vector vaccine M205 (gE ~, TK ~, gG-, El’) was 0.18 (S.E.=0.19). The transmission of vaccine strain M206 (gE - , TK- , gG ~ ), the control strain without El, was similar to the vector vaccine strain, with El. Also after inoculation with the vaccine strain M206 (gE ~, TK ~, gG -), inoculated pigs excreted the virus during a similar short period (4.4 days) with a similar average daily virus titre in OPF as pigs inoculated with the vector vaccine M205 (gE -, TK- , to the gG-, El+). None of the pigs contact-exposed vaccine strain M206 (gE _ , TK - , gG _ ) excreted the
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virus or developed neutralizing antibodies. Thus, the estimated R for the vaccine strain M206 (gE - , TK - , gG ~ ) was 0. The observed difference in reproduction ratio between the vector vaccine M205 (gE _, TK- , gG ~, El+) and the vaccine strain M206 (gE- , TK-, gG ) was not statistically significant. Transmission of co-inoculated gE-positive (Sterksel) PRV
gE-negative
(M141) and
It is possible that a transmissible gE-negative TKpositive PRV strain could arise through recombination, if wild-type PRV-infected pigs are vaccinated with a gE-negative TK-negative PRV (vector) strain. We therefore investigated whether gE-negative PRV could be transmitted in a population which is co-infected with wild-type PRV. Therefore, an experiment was performed to determine whether co-inoculation of a gEnegative strain (M141) and a gE-positive strain (Sterksel) affected the individual transmission of these strains among pigs. The results of this experiment are summarized in Table 2. The transmission of the coinoculated strains was compared with the transmission of separately inoculated strains. In the co-inoculated group, it was not possible to discriminate which virus strain induced the neutralizing antibodies. However, using an immunoperoxidase monolayer assay with a MAb that specifically recognizes gE, we could discriminate plaques caused by gE-negative virus and by gEpositive virus in OPF samples of the co-inoculated group. Therefore, for this group only the detection of virus excretion could be used for the detection of infection. All pigs of the co-inoculated and the separately inoculated groups became infected, i.e. induced neutralizing antibodies. In the co-inoculated group, we found that 3 contact-exposed pigs excreted the gE-negative virus, whereas all 5 pigs excreted the gE-positive virus. However, also after separate inoculation with the gE-negative virus, 3 contact-exposed pigs excreted the virus. Coinoculated pigs excreted less gE-negative virus than pigs inoculated with the gE-negative virus alone. The average daily excretion of gE-negative PRV by co-inoculated pigs and their contacts was, respectively, 30-fold and 150-fold lower than by pigs of the separately inoculated group. However, this difference in average daily titre was not statistically significant for the low number of 3 pigs. We could not estimate the R for the gE-negative virus after co-inoculation, because we could not detect whether this virus had induced neutralizing antibodies in 2 contact-exposed pigs that did not excrete gE-negative virus. When we assume that these 2 contact-exposed pigs were not infected by the gE-negative virus, the estimated R for co-inoculated gE-negative virus was 0.98 (S.E. 0.67). In the group of pigs that was separately inoculated with the gE-negative virus, all pigs became infected, and the estimated R was 6.7 (S.E.=5.09). The estimated Rs for the gE-negative PRV strain (M141) for the coinoculated and the separately inoculated pigs did not differ significantly (P=O.23). In addition, from day 14 by the co-inoculated pigs, and from day 15 by their contacts, only the gE-positive virus was excreted (data not shown). Thus, the co-inoculated strains did not compete in transmission, such that the transmission of one of them was prevented. However, co-inoculated pigs
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al. excreted less gE-negative virus than pigs inoculated with the gE-negative virus alone. Furthermore, the gEpositive virus was excreted for a longer period than the gE-negative virus. All contact-exposed pigs of the co-inoculated pigs excreted the gE-positive virus (Sterksel). The estimated basic ratio R was 4.43 (S.E. 3.06), whereas the estimated R for the same gE-positive virus in the separatelyinoculated group was 2.75 (SE. 1.74). These reproduction ratios do not differ significantly. Thus, these results indicate that the individual transmission of gE-positive PRV and gE-negative PRV strains were not severely affected by co-inoculation.
DISCUSSION Using our previously described experimental method, we estimated the transmission of PRV mutant strains with inactivated gE or TK. Strains with these mutations are used as vaccine strains or can arise through recombination with other PRV strains. In addition, we estimated the transmission of a PRV vector vaccine strain (gE - , TK _ , gG ~ ) that expresses the glycoprotein El of HCV. Our results indicated that inactivation of only gE or only TK is not sufficient to reduce the transmission of PRV to the R to < 1. Several observations support the contention that combined inactivation of gE and TK synergistically affects transmission of PRV. First, gE and TK have been shown to synergistically affect in viva replication of PRV”. Second, in agreement with this finding, the control vaccine strain M206, with inactivated gE, TK and glycoprotein gG (gG) did not cause contact-infections. Because so far no functions for virulence or replication have been assigned to gG”5,27*20,2’, the drastically reduced transmission of M206 (gE_, TK-, gG-) and M205 (gE_, TK-, gG_, El+) is likely due to combined lack of gE and TK expression, In addition, the vaccine strain 783 that lacks both gE and TK was not transmitted to lo-week-old pigsX6, although it was transmitted to one 4-day-old piglet”. However, these experiments were not designed to measure transmission and it was not estimated whether Rl. Moreover, because the experimental conditions differed from our studies their results cannot be directly compared with our transmission studies. Until now only limited cases of gE-negative field isolates have been reported, that may have been derived from attenuated vaccine strains”‘. Yet, the incidence of the circulation of gE-negative PRV strains in the pig population could be underestimated because these PRV strains are not recognized in the serological gE-detection test, and do not cause neurological signs of disease. Spread of gE-negative, TK-positive PRV strains will probably not hamper the eradication of PRV as long as vaccination against PRV is practised thoroughly, since even the wild-t e strains normally do not spread in vaccinated pig&‘. When vaccination is no longer applied, reactivation of latent attenuated vaccine strains (gE -) and eventual recombination of attenuated vaccine strains with wild-type PRV could cause new infections in pigs, that are not recognized by the serological gE-detection test. Thus, at the end of the PRV eradication programmes, the gE-detection test should be
accompanied by a more general test that can also detect possible gE-negative PRV infections. The PRV vector vaccine M205 (gE_, TK-, gG_, El’) expressing El of HCV, was not transmitted among susceptible pigs after intramuscular vaccination. Inoculated pigs did not excrete the virus, indicating that intramuscular vaccination is a safe application route for PRV recombinant vaccines that prevents subsequent spread among pigs. However, after intranasal inoculation with the vector vaccine, inoculated pigs excreted the virus and one contact-exposed pig became infected, i.e. produced neutralizing antibodies against the virus. The incorporated HCV El did not significantly change the transmission of the PRV vector vaccine. It is unlikely that the vector virus M205 (gE ~, TK ~, gG ~, El+) can spread or survive: the estimated R was ~1; the M205-infected contact-exposed pigs excrete detectable amounts of virus; and the R was estimated in susceptible pigs.
did
not
However, most pigs in the swine industry are vaccinated against PRV leading to reduced spread of PRV strains. Recently, non-transmissible PRV gD mutants were developed that can be used as safe vector vaccines, because they guarantee no spread. Pigs that are inoculated with these phenotypically complemented gD mutants excrete virions that are non-infectious because they lack glycoprotein gD40,4’. When a host is co-infected with two different herpesvirus strains, recombinant virus strains may arise42-44. Therefore, it is possible that a transmissible gE-negative TK-positive PRV strain could be generated if wild-type PRV-infected pigs are vaccinated with a gE-negative TK-negative PRV (vector) strain. Our experiments predicted that a gE-negative TK-positive PRV could survive in a susceptible population by transmission. We subsequently asked whether gE-negative PRV could also be transmitted in a population which is co-infected with wild-type PRV. We therefore investigated whether co-inoculation of a gE-negative strain (M141) and a gE-positive strain (Sterksel) affected the individual transmission of these strains among pigs. Although both co-inoculated PRV strains were transmitted to contactexposed pigs, the co-inoculated pigs excreted less gEnegative virus than pigs inoculated with the gE-negative virus alone. Furthermore, the gE-positive virus was excreted for a longer period than the gE-negative virus. This finding could mean that in time gE-negative viruses have less chance on survival than gE-positive viruses. Possibly, because cell-to-cell spread of gE-negative PRV is hampered45, uninfected cells of the oropharyngeal mucosa could become earlier or more easier infected by progeny virus of the gE-positive strain than by progeny virus of the gE-negative strain. In conclusion, we demonstrated that a small-scale experimental model can be used to estimate the transmission of PRV mutant strains and PRV vector vaccines under experimental conditions. The transmission in larger populations than used in the experiments here will be the same as estimated from these experiments. The size follows logically independence of R from population from the mass-action argument46 and it was also experimentally shown to be true for PRV in pigs4’. The results
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Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
of this study indicated that deletion of only gE or only TK may not be enough to prevent the spread of PRV among susceptible pigs, and that the transmission of gE-negative strain is not firmly limited by the copresence of a gE-positive strain.
19 20
21
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Kimman, T.G. Risks connected with the use of conventional and genetically engineered vaccines. Vet. Q. 1992, 15, 110-118 Mulder, W.A.M., Priem, J., Glazenburg, K.L. eta/. Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein El of hog cholera virus. J. Gen. Viral. 1994, 75, 117-124 Mulder, W.A.M., Priem, J., Pol, J.M.A. and Kimman, T.G. Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells. J. Gen. Viral. 76, 1433-l 442 Anderson, R.M. and May, R.M. Directly transmitted infectious diseases: control by vaccination. Science 1982, 215, 10531060 Anderson, R.M. and May, R.M. Vaccination and herd immunity to infectious diseases. Nature 1985, 318, 323-329 De Jong, M.C.M. and Diekmann, 0. A method to calculate-for computer-simulated-infections-the threshold value, R,, that predicts whether or not the infection will spread. Prev. Vet. Med. 1991, 12,269-285 McFerran, J.B. and Dow, C. Studies on immunisation of pigs with the Bartha strain of Aujeszky’s disease virus. Res. Vet. Sci. 1975, 19, 17-22 De Wind, N., Zijderveld, A., Glazenburg, K., Gielkens, A. and Berns, A. Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the U, region of pseudorabies virus. J. Viral 1990, 64, 4691-4696 Kimman, T.G., De Wind, N., Oei-Lie, N., Pol, J.M.A., Berns, A.J.M. and Gielkens, A.L.J. Contribution of single genes within the short region of Aujeszky’s disease virus (suid herpesvirus type 1) to virulence, pathogenesis and immunogenicity. J. Gen. Virol. 1992, 73, 243-251 Kasza, L., Shadduck, J.A. and Christofinis, G.J. Establishment, viral susceptibility, and biological characteristics of a swine kidney cell line SK-6. Res. Vet. Sci. 1971, 13, 46-51 Moormann, R.J.M., De Rover, T., Briaire, J., Peeters, B.P.H., Gielkens, A.L.J. and Van Oirschot, J.T. Inactivation of the thymidine kinase gene of a gl deletion mutant of pseudorabies virus generates a safe but highly immunogenic vaccine strain. J. Gen. Viral. 1990, 71, 5191-1595 Van Oirschot, J.T. Induction of antibodies to glycoprotein I to different doses of a mildly virulent strain of Aujeszky’s disease virus. Vet. Rec. 1988, 18, 599-603 Jacobs, L., Meloen, R.H., Gielkens, A.L.J. and Van Oirschot, J.T. Epitope analysis of glycoprotein I of pseudorabies virus. J. Gen. Viral. 1990, 71, 881-887 De Leeuw, P.W., Wijsmuller, J.M., Zantinga, J.W. and Tielen, M.J.M. lntranasal vaccination of pigs against Aujeszky’s disease. Comparison of intranasal and parental vaccination against Aujeszky’s disease in 12-week old pigs from immunized dams. Vet 0. 1982, 4, 49-56 Diekmann, 0. Heesterbeek, J.A.P. and Metz, J.A.J. On the definition and computation on the basic reproduction ratio R, in models for infectious diseases in heterogeneous populations. J. Math. Biol. 1990, 28, 365-382 Becker, N.G. Analysis of Infectious Data. Chapman and Hall, London, 1989, pp. 139-174 Thomsen, D.R., Marchioli, C.G., Yancey, R.J. and Post, L.E. Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX. J. Viral. 1987, 61, 229-232. Van Oirschot, J.T., Moormann, R.J.M., Berns, A.J.M. and Gielkens, A.L.J. Efficacy of a pseudorabies virus vaccine strain 783 that does not express thymidine kinase and glycoprotein I. Am. J. Vet. Res. 1991, 52, 1056-l 060 Van Oirschot, J.T., Terpstra, C., Moormann, R.J.M., Berns, A.J.M. and Gielkens, A.L.J. Safety of an Aujeszky’s disease vaccine based on deletion mutant strain ,783 which does not express thymidine kinase and glycoprotein I. Vet. Rec. 1990, 127,443-446 Christensen, L.S., Medveczky, I., Strandbygaard, B.S. and Pejsak, Z. Characterization of field isolates of suid herpesvirus 1 (Aujeszky’s disease virus) as derivatives of attenuated vaccine strains. Arch. Viral. 1992, 124, 225-234 Bouma, A., De Jong, M.C.M. and Kimman, T.G. Two pseudorabies virus strains, that differ in virulence and virus excretion, have equal transmission in groups of vaccinated pigs. Am. J. Vet. Res. accepted Heffner, S., Kovacs, F., Klupp, B.G. and Mettenleiter, T.C. Glycoprotein gp50-negative pseudorabies virus: a novel
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
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approach toward a nonspreading live herpesvirus vaccine. J. Virol. 1993, 67, 1529-1537 Peeters, B., Bouma, A., de Bruin, T., Moormann, R., Gielkens, A. and Kimman, T. Non-transmissible pseudorabies virus gp50 mutants: a new generation of safe live vaccines. Vaccine 1994, 12, 375-380 Henderson, L.M., Levings, R.L., Davies, A.J. and Sturtz, DR. Recombination of pseudorabies virus vaccine strains in swine. Am. J. Vet. Res. 1991, 52, 820-825 Glazenburg, K.L., Moormann, R.J.M., Kimman, T.G., Gielkens, A.L.J. and Peeters, B.P.H. Genetic recombination in pigs: evidence that homologous recombination between insert sequences is less frequent than between autologous sequences. Arch. Viral. 1995, 140, 671-685
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Dangler, C., Henderson, L., Bowman, L. and Deaver, R. Direct isolation and identification of recombinant pseudorabies virus strains from tissues of experimentally co-infected animals. Am. J. Vet Res. 1993, 54, 540-545 Jacobs, L. Glycoprotein E of pseudorabies virus and homologous proteins in other alphaherpesvirinae. Brief review. Arch. Virol. 1994, 137, 209-228 De Jong, M.C.M., Diekmann, 0. and Heesterbeek, J.A.P. How does transmission of infection depend on population size? In: Epidemic Models: their Structure and Relation to Data (Ed. Mollison, D.). Cambridge University Press, 1995, pp. 84-94 Bouma, A., De Jong, M.C.M. and Kimman, T.G. Transmission of pseudorabies virus within pig populations is independent of the size of the population. Rev. Vet. Med. 1995, 23, 163-172
Vaccine 1995 Volume 13 Number 18 1769
1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264-410X/95 $iO+O.OO
0264-410X(95)00156-5
Experimental quantification of transmission of genetically engineered pseudorabies virus W.A.M. Mulder*Q, M.C.M. De Jong*, J. Priem*, J.M.A. Pal* and T.G. Kimman*
A. Bouma*,
There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent d@erent PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PR V mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PR V vector vaccine (gE- , TK- , gG- ) that expresses the glycoprotein El (El) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when RI, the infection can spread; when R
virus mutant
strains;
experimental
transmission;
Pseudorabies virus (PRV) (synonyms: Aujeszky’s disease virus, suid herpesvirus-1) is a highly neurotropic alphaherpesvirus that causes Aujeszky’s disease in pigs’-‘. Swine are the natural host for this virus, its sole reservoir, and the sole source of virus transmission. PRV is prevalent in most parts of the world and causes economic losses in the swine industry and leads to trade barriers between countries. In the Netherlands and other countries of the European Community, PRV eradication programmes have started using marker vaccines that enable serologic differentiation between vaccinated and *Institute for Animal Science and Health, Department of Porcine and Exotic Viral Diseases and Department of Pathobiology and Epidemiology, P.O. Box 365, 8200 AJ Lelystad, The Netherlands. tuniversity of Utrecht, Department of Veterinary Pathology, Yalelaan 1, 3508 TD Utrecht, The Netherlands. $To whom all correspondence should be addressed. (Received IO February 1995; revised 30 June 1995; accepted 14 July 1995)
pigs
infected pigs4,‘. The current PRV vaccines protect against severe clinical signs of disease but usually do not prevent virus multiplication and excretion or the establishment of latency after infection6”. Vaccines against PRV should not only be applied to prevent the clinical consequences of a field infection, but first and foremost these vaccines should be applied to reduce the transmission of PRV among pigs, thus supporting the eradication of the virus’. In most eradication programmes only marker vaccines that lack expression of glycoprotein E (gE) are allowed. In addition, some vaccines lack expression of thymidine kinase (TK) to further decrease the virulence of the vaccine. Glycoprotein E promotes cellto-cell spread of the virus in vitro”“. In vivo gE is important for virulence and involved in invasion of the central nervous system”p’3. The viral TK functions in the salvage pathway for the supply of deoxynucleotides and is also important for expression of virulence14,‘5. Besides for eradication of PRV, PRV vaccines can be used as vector vaccines that express one or more
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1995 Volume
13 Number
18 1763
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
Table 1
Transmission
Experiment/PRV 1. 2. 3.
M205 M205 Ml41 M206 Ml41 M207
of intranasally
strain
(gE-, TK-, gG-, El’) (gE-, TK-, gG-, El+) (aE-) (GE-; TK-, gG-) (gE-) (TK-)
inoculated
PRV mutant strains among pigs Average duration of excretion= (days)
Average daily t&e6 (log,,, p.f.u. per g OPF)
Number of pigs with neutralizing Ab
Number of virus-excreting
in.
con.
in.
con.
in.
con.
in.
con.
5’ 5 5 5 5 5
1 0 5 0 5 5
5 :
0 0 3 0 5 5
3.2 4.0 10.4 4.4 10.2 9.8
?I 12.7 0 12.2 11.8
4.7 4.9 5.6 4.5 6.1 6.4
: 6.0 0 5.6 6.5
Z 5
pigs
aThe number of days from the first until the last day of PRV excretion averaged over those pigs that did excrete PRV on at least one day. bArithmetic average titre over the days on which the pig did excrete PRV. ‘Each of figures in this and the next three columns refer to a total of 5 pigs. in=innocukted pigs and co&contact-exposed pigs
heterologous genes encoding immunorelevant proteins of other microorganisms’6.’ . Recently, the application of PRV as an efficient vector vaccine was demonstrated by Van Zijl et al.“, who showed that immunization with PRV recombinants that expressed the envelope El (El) of hog cholera virus (HCV) protected pigs against both pseudorabies and classical swine fever. Before these live vector vaccines can be applied, potential harmful sideeffects need to be evaluated”. Previously we reported that the expression of El by PRV did not change cell or host tropism, nor did it change the virulence of either non-virulent or virulent PRV strains2’,“. In this study we used a small-scale experimental model’ to estimate the transmission among susceptible pigs of PRV mutant strains that lack gE or TK, and of the PRV vector vaccines (gE , TK ~, gG-) with or without El of HCV. The estimated number of secondary cases per infectious individual, i.e. the reproduction ratio R, was used to estimate the spread from inoculated animals to susceptible contact-exposed animals. The ratio R has a threshold property; when Rl, the infection can spread; when R< 1, the infection will disappear22~24. We subsequently investigated whether gE-negative PRV could also be transmitted in a population which is co-infected with wild-type PRV. Therefore, experiments were performed to determine whether co-inoculation of a gE-negative and a gE-positive PRV strain affected the individual transmission of these strains.
MATERIALS
AND METHODS
Virus strains
The virulent wild-type strain NIA-325 was used as a parent strain for the development of the mutant viruses. Mutant viruses were generated by overlap recombination of four to five DNA fragments together compromising the entire PRV genome and insertion mutagenesis using an oligonucleotide with translational stop codons in all reading frames and an EcoRI site as described26.27. Mutant virus strains were plaque-purified three times on SK-6 cells2s and their integrity was confirmed by restriction enzyme analysis of the linker insertion sites. The PRV sequences immediately flanking the oligonucleotide insertion sites did not contain alterations. The gE-negative PRV strain (M141) has a deletion of 1729 base pairs (bp) in the gene encoding gE. The deletion was generated between the DraI site in the 5’ end of the gE gene and the EcoRI site of an oligonucleo-
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Vaccine 1995 Volume 13 Number 18
tide insertion mutant in the 3’ end of the gene, deleting nucleotides 6833-856226. The TK-negative strain (M207) contains a deletion of 19 bp in the 3’ part of the UL23 gene encoding TK. The 19 bp deletion is identical to the deletion in the vaccine strain 78329. The strain lacks functional TK activity and is completely nonvirulent for mice. The construction and characterization of strain M205 (gE-, TK-, gG_, El’), the vector vaccine expressing El of HCV, and of the control strain M206 (gE _, TK _, gG - ) without El, has been described previously’*. The gene encoding El of HCV was inserted into the glycoprotein gG (gG) locus. Expression of glycoprotein El of HCV did not change the virulence or pathogenesis of this PRV vector virus in vivo20.
The gE-positive PRV strain (Sterksel), a mildly virulent field isolate”, was used for the co-inoculation experiment with the gE-negative strain.
Animal experiments
Dutch Landrace pigs were obtained from the specificpathogen-free (SPF) herd of the Institute for Animal Health and Science. The pigs were born to unvaccinated sows and had no antibodies against PRV at the start of the experiment. Four experiments were performed independently; the first three experiments were designed to estimate the transmission of PRV mutant strains (Table 1) and the fourth experiment was designed to investigate a possible competition in transmission between gEnegative and gE-positive PRV strains (Table 2). The transmission of the vector vaccine M205 (gE -, TK- , gG-, El’) expressing El was investigated once after intramuscular inoculation, because this route is mostly used for vaccination against PRV. In all other experiments, PRV strains were intranasally inoculated, because this represents the natural route of infection of PRV and represents the “worst-case” route by which the virus is transmitted. To investigate a possible competition in transmission (exp. 4) a gE-negative strain (M141) and a gE-positive strain (Sterksel) were coinoculated in pigs. The individual transmission of these co-inoculated strains was compared with the transmission of separately inoculated gE-negative or gE-positive PRV strains. Groups of ten 3-week-old pigs were placed in separate isolation units at a density of 0.85 m2 pig-‘. Five pigs, randomly chosen from the ten pigs in each group, were
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al. Table 2
Competition
in transmission
between
intranasally
Number of virus-excreting
pigs
co-inoculated
gE-negative
and gE-positive
PRV strains
Average duration of excretiona (days)
Average daily titreb (log,, p.f.u./g OPF)
in.
con.
in.
con.
in.
con.
Co-inoculated: Ml41 (gE-) Sterksel (gE+)
5= 5
3 5
9.2 13.6
5.3 7.0
4.6 5.5
4.3 5.8
Separately inoculated: Ml41 (gE-) Sterksel (gE+)
3 5
z
11.3 14.0
3.7 7.6
6.1 5.6
6.5 5.4
Inoculation/PRV
strain
7he number of days from the first until the last day of PRV excretion averaged over those pigs that did excrete PRV on at least one day. *Arithmetic average titre over the days on which the pig did excrete PRV. “Each of figures in this and the next three columns refer to a total of 5 pigs. in=innoc&ted pigs and co&ontact-exposeb pigs
placed in a separate cubicle. The remaining 5 pigs of each group were inoculated with 10’ plaque forming units (p.f.u.) virus in the first three experiments (Table I), or with lo5 p.f.u. virus in the competition experiments (Table 2). Virus suspension (0.5 ml) was slowly administered into each nostril during inspiration. After 24 h, pigs were reunited to their previous companions, thereby contact-exposing these other 5 pigs to a PRV mutant strain excreted by the inoculated pigs. Thus, each group consisted of 5 inoculated (possible infectious) pigs and 5 contact-exposed (susceptible) pigs. The experimental conditions (e.g. density, origin of pigs, sampling) were similar in all the experiments. Sampling procedures
Starting the day before inoculation, and then each day for 16 days after inoculation, swabs of oropharyngeal fluid (OPF) were collected from all animals to measure virus excretion. For detection of neutralizing antibodies against PRV, serum blood was taken once before inoculation, and then weekly until the end of the experiment, 5 weeks after inoculation. Swabs were used to collect OPF samples. Swabs were extracted with 4 ml Dulbecco’s modification of Eagle’s medium supplemented with antibiotics and 2% fetal calf serum as described previously, and were stored at - 70”C2’. Subsequently, OPF samples were titrated for virus by plaque assay on monolayers of SK-6 cells. In experiment 4 an immunoperoxidase monolayer assay was performed on infected monolayers using MAb 2 that detects a conformational dependent epitope on the antigen binding domain E of gE”. Therefore, in OPF samples of the co-inoculated group, we could discriminate plaques caused by gEpositive virus (Sterksel strain) and plaques caused by gE-negative virus (strain M141). Virus titres are expressed as log,, p.f.u. per g OPF. Serum samples were stored at - 70°C and were tested for virus neutralizing antibodies against PRV as described by De Leeuw et ~1.“~Detection of infectious PRV in OPF samples or induction of neutralizing antibodies against PRV was considered evidence of infection. Statistical
methods
To estimate transmission of virus within experimental groups of pigs, we used the stochastic SusceptibleInfectious-Removed (SIR) model, described elsewhere’. The extent of transmission is expressed as the reproduction ratio R, which is mathematically defined as the
average number of new infections caused by one infectious individual during its entire infectious period”. Given the outcome of the observed outbreak in a population, the reproduction ratio R can be estimated, using a martingale estimatorX4:
The variables are the total number of pigs in the population (N), the number of susceptible pigs at the start of the infection chain (So), the number of susceptible pigs at the end of the infection chain (D), and the percentage of infectivity that is left, when the last susceptible pig becomes infected (Z). When all susceptible pigs become infected (S,=O), 2 is used to make a correction, because a part of the infectivity may not have been used after the last contact-infection. 2 was estimated by the amount of excreted virus from the moment of the last contact-infection until the end of the experiment divided by the total amount of excreted virus. The threshold value R predicts whether or not the infection will spread. When Rl, the infection can spread; when R< 1, the infection will disappear22p24. The algorithm for calculating exact probabilities of the final outcomes has been described previously. The exact probability distribution for R=l was used to test whether the R was larger than one, and the distribution based on the pooled estimate was used to compare transmission between virus strains’. For multiple paired comparisons of R within experiments, we did not use P=O.O5 as a criterion for significance but P=O.Ol. Differences in duration of virus excretion and virus titres in the OPF were tested with the Mann-Whitney U test. RESULTS Transmission
of PRV mutant strains
The results of the experiments designed to estimate the transmission of PRV mutant strains that lack gE or TK, or that express El of HCV are summarized in Table I. Pigs were inoculated intranasally, because this represents the natural route of infection of PRV and represents the “worst-case” route by which the virus can be transmitted. In the experiments, induction of
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Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al.
neutralizing antibodies against PRV and virus excretion were measured, and both were used for the detection of infection. As can be seen from the results in Table I, detection of virus excretion appears less sensitive than detection of neutralizing antibodies. In some cases, pigs developed neutralizing antibodies, but virus excretion was not detected. This occurred for 1 pig contactexposed to the vector vaccine M20.5 (gE ~ , TK ~ , gG _ , El+) expressing El in experiment 1, and for 2 pigs contact-exposed to the gE-negative virus in experiment 2. Both the inoculated and contact-exposed pigs excreted the gE-negative PRV strain (M141) for a long period (lo-12 days, Table I), except the contact-exposed pigs in experiment 4 that excreted the virus for a shorter period (3.7 days, Table 2). The pooled estimate for the reproduction ratio for the three independent experiments of the gE-negative PRV strain was 10.1 (S.E. 4.98), and was significantly 1 (P=O.O005). Furthermore, in experiment 2, the reproduction ratio of this gEnegative strain was significantly larger than the R for vector vaccine strain M205 (gE_, TK-, gG_, El+) (P=O.O075). In experiment 3, the R of the gE-negative strain was significantly larger than the R of the vaccine strain M206 (gE_ , TK-, gG-) (P=O.O006); R of the gE-negative strain was not significantly larger than the R of the TK-negative PRV strain (M207) (P=O.ll). Thus, these results indicate that a gE-negative PRV strain can spread in a susceptible, i.e. sero-negative, pig population. In experiment 3, both inoculated and contact-exposed pigs excreted the TK-negative PRV strain (M207) for a similar long period as the gE-negative strain. The estimated R for the TK-negative strain was 5.0 (S.E.=3.53). In this experiment, the reproduction ratio for the TKnegative PRV strain was not significantly 1 (P=O.O813). The R of the TK-negative strain was not significantly larger than the R of the vaccine strain M206 (gE -, TK - , gG ~ ) (P=O.O374). In experiment 1, contact-exposed pigs were not infected after intramuscular inoculation of pigs with the PRV vector vaccine M205 (gE _, TK _, gG _, El+) (data not shown). The intramuscular inoculated pigs developed neutralizing antibodies, but did not excrete the virus (data not shown). So, the estimated R for the vector vaccine M205 (gE ~, TK ~, gG _, El+) after intramuscular inoculation was 0. In contrast to the intramuscular route, intranasally inoculated pigs excreted the vector vaccine in both experiments 1 and 2. The period of virus excretion was short; on average, respectively, 3.2 and 4.0 days. None of the contactexposed pigs excreted virus, but 1 contact-exposed pig in experiment 1 developed neutralizing antibodies. The pooled estimate for the reproduction ratio for the intranasally inoculated vector vaccine M205 (gE ~, TK ~, gG-, El’) was 0.18 (S.E.=0.19). The transmission of vaccine strain M206 (gE - , TK- , gG ~ ), the control strain without El, was similar to the vector vaccine strain, with El. Also after inoculation with the vaccine strain M206 (gE ~, TK ~, gG -), inoculated pigs excreted the virus during a similar short period (4.4 days) with a similar average daily virus titre in OPF as pigs inoculated with the vector vaccine M205 (gE -, TK- , to the gG-, El+). None of the pigs contact-exposed vaccine strain M206 (gE _ , TK - , gG _ ) excreted the
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virus or developed neutralizing antibodies. Thus, the estimated R for the vaccine strain M206 (gE - , TK - , gG ~ ) was 0. The observed difference in reproduction ratio between the vector vaccine M205 (gE _, TK- , gG ~, El+) and the vaccine strain M206 (gE- , TK-, gG ) was not statistically significant. Transmission of co-inoculated gE-positive (Sterksel) PRV
gE-negative
(M141) and
It is possible that a transmissible gE-negative TKpositive PRV strain could arise through recombination, if wild-type PRV-infected pigs are vaccinated with a gE-negative TK-negative PRV (vector) strain. We therefore investigated whether gE-negative PRV could be transmitted in a population which is co-infected with wild-type PRV. Therefore, an experiment was performed to determine whether co-inoculation of a gEnegative strain (M141) and a gE-positive strain (Sterksel) affected the individual transmission of these strains among pigs. The results of this experiment are summarized in Table 2. The transmission of the coinoculated strains was compared with the transmission of separately inoculated strains. In the co-inoculated group, it was not possible to discriminate which virus strain induced the neutralizing antibodies. However, using an immunoperoxidase monolayer assay with a MAb that specifically recognizes gE, we could discriminate plaques caused by gE-negative virus and by gEpositive virus in OPF samples of the co-inoculated group. Therefore, for this group only the detection of virus excretion could be used for the detection of infection. All pigs of the co-inoculated and the separately inoculated groups became infected, i.e. induced neutralizing antibodies. In the co-inoculated group, we found that 3 contact-exposed pigs excreted the gE-negative virus, whereas all 5 pigs excreted the gE-positive virus. However, also after separate inoculation with the gE-negative virus, 3 contact-exposed pigs excreted the virus. Coinoculated pigs excreted less gE-negative virus than pigs inoculated with the gE-negative virus alone. The average daily excretion of gE-negative PRV by co-inoculated pigs and their contacts was, respectively, 30-fold and 150-fold lower than by pigs of the separately inoculated group. However, this difference in average daily titre was not statistically significant for the low number of 3 pigs. We could not estimate the R for the gE-negative virus after co-inoculation, because we could not detect whether this virus had induced neutralizing antibodies in 2 contact-exposed pigs that did not excrete gE-negative virus. When we assume that these 2 contact-exposed pigs were not infected by the gE-negative virus, the estimated R for co-inoculated gE-negative virus was 0.98 (S.E. 0.67). In the group of pigs that was separately inoculated with the gE-negative virus, all pigs became infected, and the estimated R was 6.7 (S.E.=5.09). The estimated Rs for the gE-negative PRV strain (M141) for the coinoculated and the separately inoculated pigs did not differ significantly (P=O.23). In addition, from day 14 by the co-inoculated pigs, and from day 15 by their contacts, only the gE-positive virus was excreted (data not shown). Thus, the co-inoculated strains did not compete in transmission, such that the transmission of one of them was prevented. However, co-inoculated pigs
Transmission of PRV mutant strains among pigs: W.A.M. Mulder et al. excreted less gE-negative virus than pigs inoculated with the gE-negative virus alone. Furthermore, the gEpositive virus was excreted for a longer period than the gE-negative virus. All contact-exposed pigs of the co-inoculated pigs excreted the gE-positive virus (Sterksel). The estimated basic ratio R was 4.43 (S.E. 3.06), whereas the estimated R for the same gE-positive virus in the separatelyinoculated group was 2.75 (SE. 1.74). These reproduction ratios do not differ significantly. Thus, these results indicate that the individual transmission of gE-positive PRV and gE-negative PRV strains were not severely affected by co-inoculation.
DISCUSSION Using our previously described experimental method, we estimated the transmission of PRV mutant strains with inactivated gE or TK. Strains with these mutations are used as vaccine strains or can arise through recombination with other PRV strains. In addition, we estimated the transmission of a PRV vector vaccine strain (gE - , TK _ , gG ~ ) that expresses the glycoprotein El of HCV. Our results indicated that inactivation of only gE or only TK is not sufficient to reduce the transmission of PRV to the R to < 1. Several observations support the contention that combined inactivation of gE and TK synergistically affects transmission of PRV. First, gE and TK have been shown to synergistically affect in viva replication of PRV”. Second, in agreement with this finding, the control vaccine strain M206, with inactivated gE, TK and glycoprotein gG (gG) did not cause contact-infections. Because so far no functions for virulence or replication have been assigned to gG”5,27*20,2’, the drastically reduced transmission of M206 (gE_, TK-, gG-) and M205 (gE_, TK-, gG_, El+) is likely due to combined lack of gE and TK expression, In addition, the vaccine strain 783 that lacks both gE and TK was not transmitted to lo-week-old pigsX6, although it was transmitted to one 4-day-old piglet”. However, these experiments were not designed to measure transmission and it was not estimated whether Rl. Moreover, because the experimental conditions differed from our studies their results cannot be directly compared with our transmission studies. Until now only limited cases of gE-negative field isolates have been reported, that may have been derived from attenuated vaccine strains”‘. Yet, the incidence of the circulation of gE-negative PRV strains in the pig population could be underestimated because these PRV strains are not recognized in the serological gE-detection test, and do not cause neurological signs of disease. Spread of gE-negative, TK-positive PRV strains will probably not hamper the eradication of PRV as long as vaccination against PRV is practised thoroughly, since even the wild-t e strains normally do not spread in vaccinated pig&‘. When vaccination is no longer applied, reactivation of latent attenuated vaccine strains (gE -) and eventual recombination of attenuated vaccine strains with wild-type PRV could cause new infections in pigs, that are not recognized by the serological gE-detection test. Thus, at the end of the PRV eradication programmes, the gE-detection test should be
accompanied by a more general test that can also detect possible gE-negative PRV infections. The PRV vector vaccine M205 (gE_, TK-, gG_, El’) expressing El of HCV, was not transmitted among susceptible pigs after intramuscular vaccination. Inoculated pigs did not excrete the virus, indicating that intramuscular vaccination is a safe application route for PRV recombinant vaccines that prevents subsequent spread among pigs. However, after intranasal inoculation with the vector vaccine, inoculated pigs excreted the virus and one contact-exposed pig became infected, i.e. produced neutralizing antibodies against the virus. The incorporated HCV El did not significantly change the transmission of the PRV vector vaccine. It is unlikely that the vector virus M205 (gE ~, TK ~, gG ~, El+) can spread or survive: the estimated R was ~1; the M205-infected contact-exposed pigs excrete detectable amounts of virus; and the R was estimated in susceptible pigs.
did
not
However, most pigs in the swine industry are vaccinated against PRV leading to reduced spread of PRV strains. Recently, non-transmissible PRV gD mutants were developed that can be used as safe vector vaccines, because they guarantee no spread. Pigs that are inoculated with these phenotypically complemented gD mutants excrete virions that are non-infectious because they lack glycoprotein gD40,4’. When a host is co-infected with two different herpesvirus strains, recombinant virus strains may arise42-44. Therefore, it is possible that a transmissible gE-negative TK-positive PRV strain could be generated if wild-type PRV-infected pigs are vaccinated with a gE-negative TK-negative PRV (vector) strain. Our experiments predicted that a gE-negative TK-positive PRV could survive in a susceptible population by transmission. We subsequently asked whether gE-negative PRV could also be transmitted in a population which is co-infected with wild-type PRV. We therefore investigated whether co-inoculation of a gE-negative strain (M141) and a gE-positive strain (Sterksel) affected the individual transmission of these strains among pigs. Although both co-inoculated PRV strains were transmitted to contactexposed pigs, the co-inoculated pigs excreted less gEnegative virus than pigs inoculated with the gE-negative virus alone. Furthermore, the gE-positive virus was excreted for a longer period than the gE-negative virus. This finding could mean that in time gE-negative viruses have less chance on survival than gE-positive viruses. Possibly, because cell-to-cell spread of gE-negative PRV is hampered45, uninfected cells of the oropharyngeal mucosa could become earlier or more easier infected by progeny virus of the gE-positive strain than by progeny virus of the gE-negative strain. In conclusion, we demonstrated that a small-scale experimental model can be used to estimate the transmission of PRV mutant strains and PRV vector vaccines under experimental conditions. The transmission in larger populations than used in the experiments here will be the same as estimated from these experiments. The size follows logically independence of R from population from the mass-action argument46 and it was also experimentally shown to be true for PRV in pigs4’. The results
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of this study indicated that deletion of only gE or only TK may not be enough to prevent the spread of PRV among susceptible pigs, and that the transmission of gE-negative strain is not firmly limited by the copresence of a gE-positive strain.
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Gustafson, D.P. Pseudorabies. In: Diseases of Swine (Eds Leman, A.D., Straw, B., Glock, RD., Mengeling, W.L., Penny, R.H.C. and Scholl, E.). Iowa State University Press, Ames, 1986, pp. 274-289 Pensaert, M.B. and Kluge, J.P. Pseudorabies virus (Aujeszky’s disease). In: Virus Infections of Porcines (Ed. Pensaert, M.B.). Elsevier Science Publishers BV, Amsterdam, 1989, pp. 39-64 Wittmann, G. and Rziha, H.-J. Aujeszky’s disease (pseudorabies) in pigs. In: Herpesvirus Diseases of Cattle, Horses, and Pig (Ed. Wittmann, G.). Kluwer Academic Publishers, Nor-well, Massachusetts, 1989, pp. 230-325 Van Oirschot, J.T. Vaccination in food animals. Vaccine 1994, 12,415-418 Van Oirschot, J.T., Rziha, H.-J., Moonen, P.J.L.M., Pol, J.M.A. and Van Zaane, D. Differentiation of serum antibodies from pigs vaccinated or infected with Aujeszky’s disease virus by a competitive enzyme immunoassay. J. Gen. Viral. 1986, 67, 1179-1182 Kimman, T.G. Acceptability of Aujeszky’s disease vaccines. Dev. Viol. Standard 1992, 79, 129-136 Pensaert, M., Gielkens, A.L.J., Lomniczi, B., Kimman, T.G., Vannier, P. and Eloit, M. Round table on control of Aujeszky’s disease and vaccine development based on molecular biology. Vet. Microbial. 1992, 33, 53-67 De Jong, M.C.M. and Kimman, T.G. Experimental quantification of vaccine-induced reduction in virus transmission. Vaccine 1994, 12, 761-766 Zsak, L., Zuckermann, F., Sugg, N. and Ben-Porat, T. Glycoprotein gl of pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission. J. Viral. 1992, 66, 2316-2325 Jacobs, L., Rziha, H-J., Kimman, T.G., Gielkens, A.L.J. and Van Oirschot, J.T. Deleting valine-125 and cysteine-126 in glycoprotein I of pseudorabies virus strain NIA-3 decreases plaque size and reduces virulence in mice. Arch. Viral. 1993, 131, 251-264 Mulder, W.A.M., Jacobs, L., Priem, J. et al. Glycoprotein E-negative pseudorabies virus has reduced capability to infect second- and third-order neurons of the olfactory and trigeminal routes in the porcine central nervous system. J. Gen. Viral. 1994, 75, 3095-3106 Kritas, S.K., Pensaert, M.B. and Mettenleiter, T.C. Invasion and spread of single glycoprotein deleted mutants of Aujeszky’s disease virus (ADV) in the trigeminal nervous pathways of pigs after intranasal inoculation. Vet. Microbial. 1994, 40, 323-334 Kritas, SK., Pensaert, M.B. and Mettenleiter, T.C. Role of envelope glycoproteins gl, gp63, and glll in the invasion and spread of Aujeszky’s disease virus in the olfactory nervous pathway of the pig. J. Gen. Viral. 1994, 75, 2319-2327 Roizman, B. and Pears, A.E. In: Virology (Eds Fields, B.N., Knipe, D.M., Chanock, R.M., Hirsch, MS., Melnick, L. and Monath, T.P.). Raven, New York, 1990, pp. 1795-1841 Kimman, T.G., De Wind, N., De Bruin, T., De Visser, Y. and Voermans, J. Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity. Virology 1994, 205, 51 l-51 8 Thomsen, D.R., Marotti, K.R., Palermo, D.P., and Post, L.E. Pseudorabies virus as a live vector for expression of foreign genes. Gene 1987, 57, 261-265 Whealy, M.E., Baumeister, K., Robbins, A.K. and Enquist, L.W. A herpesvirus vector for expression of glycosylated membrane antigens: fusion proteins of pseudorabies virus glll and human immunodeficiency virus type 1 envelope glycoproteins. J. Viral. 1988, 62, 4185-4194 Van Zijl, M., Wensvoort, G., De Kluyver, E. et al. Live attenuated pseudorabies virus expressing envelope glycoprotein El of hog cholera virus protects swine both against pseudorabies and hog cholera. J. Viral. 1991, 65, 2761-2765
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approach toward a nonspreading live herpesvirus vaccine. J. Virol. 1993, 67, 1529-1537 Peeters, B., Bouma, A., de Bruin, T., Moormann, R., Gielkens, A. and Kimman, T. Non-transmissible pseudorabies virus gp50 mutants: a new generation of safe live vaccines. Vaccine 1994, 12, 375-380 Henderson, L.M., Levings, R.L., Davies, A.J. and Sturtz, DR. Recombination of pseudorabies virus vaccine strains in swine. Am. J. Vet. Res. 1991, 52, 820-825 Glazenburg, K.L., Moormann, R.J.M., Kimman, T.G., Gielkens, A.L.J. and Peeters, B.P.H. Genetic recombination in pigs: evidence that homologous recombination between insert sequences is less frequent than between autologous sequences. Arch. Viral. 1995, 140, 671-685
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Dangler, C., Henderson, L., Bowman, L. and Deaver, R. Direct isolation and identification of recombinant pseudorabies virus strains from tissues of experimentally co-infected animals. Am. J. Vet Res. 1993, 54, 540-545 Jacobs, L. Glycoprotein E of pseudorabies virus and homologous proteins in other alphaherpesvirinae. Brief review. Arch. Virol. 1994, 137, 209-228 De Jong, M.C.M., Diekmann, 0. and Heesterbeek, J.A.P. How does transmission of infection depend on population size? In: Epidemic Models: their Structure and Relation to Data (Ed. Mollison, D.). Cambridge University Press, 1995, pp. 84-94 Bouma, A., De Jong, M.C.M. and Kimman, T.G. Transmission of pseudorabies virus within pig populations is independent of the size of the population. Rev. Vet. Med. 1995, 23, 163-172
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