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Changing receptor use of PRRSV leads to different virological, immunological and clinical outcome: impact on diagnosis and control

Hans Nauwynck (UGent) , Ilias Frydas (UGent) , Isaura Christiaens (UGent) , Wander Van Breedam (UGent) , Caroline Bonckaert (UGent) and Ivan Trus (UGent)
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Abstract
A new disease characterized by reproductive and respiratory problems emerged in Northern America and Western Europe in the late eighties, early nineties. It was caused by a porcine arterivirus, which based on the symptoms was called porcine reproductive and respiratory syndrome virus (PRRSV)(Meulenberg et al., 1993). On the two continents, two clearly different genetic/antigenic viruses were circulating: an American type (amPRRSV) and a European type (euPRRSV). Based on serological examinations, it was shown that amPRRSV circulated already earlier in Northern America and by genetic analysis, more genetic variation was detected for amPRRSV in Northern America than for euPRRSV in Western Europe. This latter finding could be attributed to the earlier circulation and/or multiple introductions of the virus in the American pig population. Western Europe was confronted with a single introduction, starting in Western Germany. As a consequence, the early euPRRSV strains were genetically closely related (Stadejek et al., 2002). In the early nineties, amPRRSV was proven to be more pathogenic than euPRRSV. Indeed, whereas both virus types had the same power to give reproductive problems upon infection during late gestation, amPRRSV was giving more general clinical signs (fever, anorexia) and respiratory problems than euPRRSV. Only in combinations with other pathogens/toxins euPRRSV was able to induce overt general and respiratory clinical signs (Van Reeth et al., 1996). By recombination and genetic drift, American strains evolved fast, giving rise to new strains that were even more virulent and extremely difficult to control by commercial vaccines (atypical PRRSV, Sow Abortion and Mortality Syndrome (SAMS))(Mengeling et al., 1998). In 2006, extremely aggressive variants of amPRRSV appeared in China, which are now damaging the whole Asian pig population (long lasting high fever, respiratory and reproductive problems, high mortality) and which represent a real threat to other continents (Tian et al., 2007). In Eastern Europe, a surprisingly large variation was found for euPRRSV isolates leading to the identification of new subtypes (2, 3 and 4) that were quite different from subtype 1 (Stadejek et al., 2006). At present, it is hypothesized that euPRRSV was circulating in Eastern Europe a long time before the entrance of subtype 1 in Western Europe. When the whole group of Western and Eastern European euPRRSV strains are considered, a genetic variation of euPRRSV was found that was even larger than the one found with amPRRSV in the US, leading to a very complex genetic world picture of PRRSV. Eastern European PRRSV strains of subtype 2 (prototype Bor) and 3 (prototype Lena) have been shown to be more virulent than the Western European strains (subtype 1 (prototype Lelystad))(Karniychuk et al., 2009). The euPRRSV strain Lena is even as virulent and pathogenic as the high fever disease amPRRSV in Asia. Over the years in Western Europe, euPRRSV remained mainly linked with reproductive problems. Fever and respiratory problems were absent upon experimental single inoculations. However, starting from mid 2013, PRRSV is responsible for flu-like problems in nurseries in Belgium and most probably also neighboring countries (unpublished data). Upon experimental inoculation with one of these isolates, Flanders 13, fever and respiratory problems were reproduced. Genetically, this virus is quite different from other circulating PRRS viruses. The pathogenesis of PRRSV is fully determined by differentiated macrophages. During the past 20 years, the euPRRSV strains of subtype 1 (prototype Lelystad) replicated very similarly in a pig (Duan et al., 1993). Targets are differentiated macrophages that are carrying the sialoadhesin receptor (Duan et al., Vanderheijden et al., 2010, Karniychuk et al., 2013). These cells can easily be found in tonsils and lungs, lymph nodes, spleen, maternal endometrium and fetal placenta and at lower levels in all other tissues of the pig (Karniychuk et al. 2009). Because the virus does not replicate well in the upper respiratory tract, the virus is difficult to isolate from nasal swabs and the virus does not spread fast between pigs (Albina, 1997). Due to the rather restricted number of differentiated cells that are infected, virus titers of 102-104 TCID50/ml are generally found in serum. EuPRRSV strains of subtype 3 (prototype Lena) differ from LV-like strains because they are able to infect a new subset of differentiated macrophages that do not possess the sialoadhesin receptor (Frydas et al. 2013). An additional receptor is most probably responsible for this. By experiments in nasal mucosa explants it was found that the additional subset is present at high concentrations in and under the respiratory epithelial cells of the upper respiratory tract, allowing a much stronger replication in respiratory tissues (up to 10-100x higher) and giving rise to a strong viral shedding and a fulminant viremia (100x higher; virus titers up to 104-106 TCID50/ml). Based on the localisation of this new subset of susceptible macrophages, it is hypothesized that they represent nasal macrophages. These cells are forming a dense network and are taking care of the first line of defense against pathogens (Vareille et al., 2011). Destroying both nasal and alveolar lung macrophages is most probably the reason why euPRRSV Lena has been associated with secondary bacterial infections and sepsis (Karniychuk et al., 2010). The new Flanders 13-like strains are euPRRSV subtype 1 strains that are also evolving in the same direction as Lena. By using the nasal mucosa explants, this virus also replicated in non-sialoadhesin positive macrophages. The virus titers in nasal secretions from euPRRSV infected animals are in line with the replication of the virus in nasal mucosa explants. Whereas it is difficult to detect LV-like euPRRSV subtype 1 in nasal secretions, it is very easy to do so with the more virulent euPRRSV subtype 3 and Flanders 13-like strains. Transmission experiments with these different strains are ongoing in order to find out if the power of the virus to replicate in the nasal macrophages may be related to its aerogenic spread. The increase of macrophage subsets that are infected with euPRRSV in time is a dangerous evolution. A ten- to hundredfold increase of replication gives rise to viral mutants with the same magnitude. Knowing that mutagenesis is helping the virus to escape from immunity and is increasing the risk that highly virulent strains emerge is bringing Europe in a very dangerous situation. In addition, because of the close relationship of the porcine receptors sialoadhesin and CD163 with their human homologs, one should consider the risk of a species jump to humans (Van Breedam et al., 2013). With these interesting findings with euPRRSV strains, we have recently performed experiments with amPRRSV strains in nasal mucosa explants. It was found that both old (VR2332) and more recent amPRRSV strains (SDSU-73, NADC, MN-184) from the US easily replicate to high levels in both sialoadhesin positive and sialoadhesin negative macrophages in the nasal mucosa (unpublished data). This finding is explaining several things. AmPRRSV behaved differently from euPRRSV from the very beginning; it replicated in more subsets of macrophages than the old subtype 1 euPRRSV LV-like strains. Its high replication in the upper respiratory tract differs from the low level of replication of the old subtype 1 euPRRSV strains. This also explains why it was and still is very easy for amPRRSV to spread via airborne transmission (Otake et al., 2010). In addition, the higher number of macrophage subsets that are infected with amPRRSV explains why it is more virulent/pathogenic than the old subtype 1 euPRRSV strains and give more rise to secondary infections. It also explains why amPRRSV is able to replicate in sialoadhesin-negative pigs (Prather et al., 2013). All these findings are very important in function of the diagnosis. An etiological diagnosis of PRRS during reproductive failure is straightforward for all PRRSV strains. PRRSV is replicating in the macrophages of the fetal placenta when they become sialoadhesin positive at late gestation (Karniychuk et al., 2009). This results in a severe placentitis and viral spread to the fetus. The placentitis is the main cause of fetal pathology and death (Karniychuk et al., 2013). Due to the huge size of the placenta and the regularly localized PRRSV replication, it is difficult to make the diagnosis from placental tissues (which part to take?). Because fetuses do not have the time to develop antibodies before they die, it is impossible to diagnose PRRS by serological examinations on fetal fluids. Taking all these pathogenetic aspects into account, it is advised to do the diagnosis of PRRS during reproductive problems by qRT-PCR on umbilical cords (connected with fetal placenta) and organ pools (lungs/spleen) of aborted fetuses. Diagnosis of euPRRS in the context of respiratory problems is very difficult with LV-like euPRRSV strains. They are always causing long-lasting infections in young piglets, independent of their health status (diseased or healthy). Therefore, what is the meaning of a positive result (demonstrating the presence of the virus in lungs or blood). In the same context, it is difficult to interpret a seroconversion. It is not because an animal is seroconverting to LV-like euPRRSV strains that the virus is responsible for problems. This diagnostic problem is one of the main reasons why most farmers did not vaccinate their piglets up till now in Western Europe. However, in pigs with flu-like problems caused by amPRRSV, subtype 2 and 3 euPRRSV and the new Flanders 13-like euPRRSV, it is easy to demonstrate the high replication of PRRSV in the upper respiratory tract by taking nasal swabs and blood and quantitating the high viral load (up to 105 TCID50/ml in nasal secretions and 104-6 TCID50/ml in blood) by virus titration or qRT-PCR. PRRSV is a difficult target for the immunity. Several branches of the immunity have been shown not to be induced or not to be functional. Low levels of interferons are induced (Van Reeth et al., 1999). Antibodies are raised starting from 8 days post infection, but it takes several weeks before a weak neutralization can be demonstrated (Labarque et al., 2000). Natural killer cells and cytotoxic T-lymphocytes are not sufficiently effective (Cao et al., 2013; Costers et al., 2009). Only neutralizing antibodies, which appear after one month and at low levels together with a not yet identified porcine killer cell are the two branches that still can do the job and should be activated by vaccination. The drift of the virus makes it a moving target and complicates the whole vaccination strategy (Labarque et al, 2004). In the near future, it is important to have access to vaccines that are adaptable, enclosing strains that are closely related to the strains circulating in the field (Nauwynck et al., 2012). The technologies for making effective adaptable inactivated (Geldhof et al., 2012) and attenuated vaccines (unpublished data) became available and should be urgently implemented in the field. The ultimate dream is the development of an adaptable marker vector vaccine (cassette system) that induces a local immunity in the respiratory tract. As long as PRRSV does not persist such as its sister-arterivirus, lactate dehydrogenase elevating virus (LDV), we should keep on going with the investment to develop vaccines. In conclusion, it is extremely important to better control PRRS in the near future and not to wait till a complete catastrophe occurs. There is an urgent need for adaptable inactivated and attenuated marker vaccines and improved biosafety measures in order to fully control PRRSV circulation. Pig producers, PRRS researchers and pharmaceutical companies should take their responsibility and join forces to come up with solutions to eradicate this ever-changing enemy that may turn into a real nightmare. In this context, funding PRRS research should be prioritized by all agencies all over the world. Not doing this is an unforgivable error not only for animal health but possibly also for human health.
Keywords
viremia, immunity, PRRSV, PRRS, arterivirus, Nidovirales, subtype, Type

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Chicago
Nauwynck, Hans, Ilias Frydas, Isaura Christiaens, Wander Van Breedam, Caroline Bonckaert, and Ivan Trus. 2014. “Changing Receptor Use of PRRSV Leads to Different Virological, Immunological and Clinical Outcome: Impact on Diagnosis and Control.” In Proceedings of the 23rd International Pig Veterinary Society (IPVS) Congress, Vol. 1, 23–26. International Pig Veterinary Society (IPVS).
APA
Nauwynck, H., Frydas, I., Christiaens, I., Van Breedam, W., Bonckaert, C., & Trus, I. (2014). Changing receptor use of PRRSV leads to different virological, immunological and clinical outcome: impact on diagnosis and control. Proceedings of the 23rd International Pig Veterinary Society (IPVS) congress, vol. 1 (pp. 23–26). Presented at the 23rd International Pig Veterinary Society congress (IPVS 2014), International Pig Veterinary Society (IPVS).
Vancouver
1.
Nauwynck H, Frydas I, Christiaens I, Van Breedam W, Bonckaert C, Trus I. Changing receptor use of PRRSV leads to different virological, immunological and clinical outcome: impact on diagnosis and control. Proceedings of the 23rd International Pig Veterinary Society (IPVS) congress, vol. 1. International Pig Veterinary Society (IPVS); 2014. p. 23–6.
MLA
Nauwynck, Hans, Ilias Frydas, Isaura Christiaens, et al. “Changing Receptor Use of PRRSV Leads to Different Virological, Immunological and Clinical Outcome: Impact on Diagnosis and Control.” Proceedings of the 23rd International Pig Veterinary Society (IPVS) Congress, Vol. 1. International Pig Veterinary Society (IPVS), 2014. 23–26. Print.
@inproceedings{6851166,
  abstract     = {A new disease characterized by reproductive and respiratory problems emerged in Northern America and Western Europe in the late eighties, early nineties. It was caused by a porcine arterivirus, which based on the symptoms was called porcine reproductive and respiratory syndrome virus (PRRSV)(Meulenberg et al., 1993). On the two continents, two clearly different genetic/antigenic viruses were circulating: an American type (amPRRSV) and a European type (euPRRSV). Based on serological examinations, it was shown that amPRRSV circulated already earlier in Northern America and by genetic analysis, more genetic variation was detected for amPRRSV in Northern America than for euPRRSV in Western Europe. This latter finding could be attributed to the earlier circulation and/or multiple introductions of the virus in the American pig population. Western Europe was confronted with a single introduction, starting in Western Germany. As a consequence, the early euPRRSV strains were genetically closely related (Stadejek et al., 2002). In the early nineties, amPRRSV was proven to be more pathogenic than euPRRSV. Indeed, whereas both virus types had the same power to give reproductive problems upon infection during late gestation, amPRRSV was giving more general clinical signs (fever, anorexia) and respiratory problems than euPRRSV. Only in combinations with other pathogens/toxins euPRRSV was able to induce overt general and respiratory clinical signs (Van Reeth et al., 1996). By recombination and genetic drift, American strains evolved fast, giving rise to new strains that were even more virulent and extremely difficult to control by commercial vaccines (atypical PRRSV, Sow Abortion and Mortality Syndrome (SAMS))(Mengeling et al., 1998). In 2006, extremely aggressive variants of amPRRSV appeared in China, which are now damaging the whole Asian pig population (long lasting high fever, respiratory and reproductive problems, high mortality) and which represent a real threat to other continents (Tian et al., 2007). In Eastern Europe, a surprisingly large variation was found for euPRRSV isolates leading to the identification of new subtypes (2, 3 and 4) that were quite different from subtype 1 (Stadejek et al., 2006). At present, it is hypothesized that euPRRSV was circulating in Eastern Europe a long time before the entrance of subtype 1 in Western Europe. When the whole group of Western and Eastern European euPRRSV strains are considered, a genetic variation of euPRRSV was found that was even larger than the one found with amPRRSV in the US, leading to a very complex genetic world picture of PRRSV. Eastern European PRRSV strains of subtype 2 (prototype Bor) and 3 (prototype Lena) have been shown to be more virulent than the Western European strains (subtype 1 (prototype Lelystad))(Karniychuk et al., 2009). The euPRRSV strain Lena is even as virulent and pathogenic as the high fever disease amPRRSV in Asia. Over the years in Western Europe, euPRRSV remained mainly linked with reproductive problems. Fever and respiratory problems were absent upon experimental single inoculations. However, starting from mid 2013, PRRSV is responsible for flu-like problems in nurseries in Belgium and most probably also neighboring countries (unpublished data). Upon experimental inoculation with one of these isolates, Flanders 13, fever and respiratory problems were reproduced. Genetically, this virus is quite different from other circulating PRRS viruses.

The pathogenesis of PRRSV is fully determined by differentiated macrophages. During the past 20 years, the euPRRSV strains of subtype 1 (prototype Lelystad) replicated very similarly in a pig (Duan et al., 1993). Targets are differentiated macrophages that are carrying the sialoadhesin receptor (Duan et al., Vanderheijden et al., 2010, Karniychuk et al., 2013). These cells can easily be found in tonsils and lungs, lymph nodes, spleen, maternal endometrium and fetal placenta and at lower levels in all other tissues of the pig (Karniychuk et al. 2009). Because the virus does not replicate well in the upper respiratory tract, the virus is difficult to isolate from nasal swabs and the virus does not spread fast between pigs (Albina, 1997). Due to the rather restricted number of differentiated cells that are infected, virus titers of 102-104 TCID50/ml are generally found in serum. EuPRRSV strains of subtype 3 (prototype Lena) differ from LV-like strains because they are able to infect a new subset of differentiated macrophages that do not possess the sialoadhesin receptor (Frydas et al. 2013). An additional receptor is most probably responsible for this. By experiments in nasal mucosa explants it was found that the additional subset is present at high concentrations in and under the respiratory epithelial cells of the upper respiratory tract, allowing a much stronger replication in respiratory tissues (up to 10-100x higher) and giving rise to a strong viral shedding and a fulminant viremia (100x higher; virus titers up to 104-106 TCID50/ml). Based on the localisation of this new subset of susceptible macrophages, it is hypothesized that they represent nasal macrophages. These cells are forming a dense network and are taking care of the first line of defense against pathogens (Vareille et al., 2011). Destroying both nasal and alveolar lung macrophages is most probably the reason why euPRRSV Lena has been associated with secondary bacterial infections and sepsis (Karniychuk et al., 2010). The new Flanders 13-like strains are euPRRSV subtype 1 strains that are also evolving in the same direction as Lena. By using the nasal mucosa explants, this virus also replicated in non-sialoadhesin positive macrophages. The virus titers in nasal secretions from euPRRSV infected animals are in line with the replication of the virus in nasal mucosa explants. Whereas it is difficult to detect LV-like euPRRSV subtype 1 in nasal secretions, it is very easy to do so with the more virulent euPRRSV subtype 3 and Flanders 13-like strains. Transmission experiments with these different strains are ongoing in order to find out if the power of the virus to replicate in the nasal macrophages may be related to its aerogenic spread. The increase of macrophage subsets that are infected with euPRRSV in time is a dangerous evolution. A ten- to hundredfold increase of replication gives rise to viral mutants with the same magnitude. Knowing that mutagenesis is helping the virus to escape from immunity and is increasing the risk that highly virulent strains emerge is bringing Europe in a very dangerous situation. In addition, because of the close relationship of the porcine receptors sialoadhesin and CD163 with their human homologs, one should consider the risk of a species jump to humans (Van Breedam et al., 2013).

With these interesting findings with euPRRSV strains, we have recently performed experiments with amPRRSV strains in nasal mucosa explants. It was found that both old (VR2332) and more recent amPRRSV strains (SDSU-73, NADC, MN-184) from the US easily replicate to high levels in both sialoadhesin positive and sialoadhesin negative macrophages in the nasal mucosa (unpublished data). This finding is explaining several things. AmPRRSV behaved differently from euPRRSV from the very beginning; it replicated in more subsets of macrophages than the old subtype 1 euPRRSV LV-like strains. Its high replication in the upper respiratory tract differs from the low level of replication of the old subtype 1 euPRRSV strains. This also explains why it was and still is very easy for amPRRSV to spread via airborne transmission (Otake et al., 2010). In addition, the higher number of macrophage subsets that are infected with amPRRSV explains why it is more virulent/pathogenic than the old subtype 1 euPRRSV strains and give more rise to secondary infections. It also explains why amPRRSV is able to replicate in sialoadhesin-negative pigs (Prather et al., 2013).

All these findings are very important in function of the diagnosis. An etiological diagnosis of PRRS during reproductive failure is straightforward for all PRRSV strains. PRRSV is replicating in the macrophages of the fetal placenta when they become sialoadhesin positive at late gestation (Karniychuk et al., 2009). This results in a severe placentitis and viral spread to the fetus. The placentitis is the main cause of fetal pathology and death (Karniychuk et al., 2013). Due to the huge size of the placenta and the regularly localized PRRSV replication, it is difficult to make the diagnosis from placental tissues (which part to take?). Because fetuses do not have the time to develop antibodies before they die, it is impossible to diagnose PRRS by serological examinations on fetal fluids. Taking all these pathogenetic aspects into account, it is advised to do the diagnosis of PRRS during reproductive problems by qRT-PCR on umbilical cords (connected with fetal placenta) and organ pools (lungs/spleen) of aborted fetuses. Diagnosis of euPRRS in the context of respiratory problems is very difficult with LV-like euPRRSV strains. They are always causing long-lasting infections in young piglets, independent of their health status (diseased or healthy). Therefore, what is the meaning of a positive result (demonstrating the presence of the virus in lungs or blood). In the same context, it is difficult to interpret a seroconversion. It is not because an animal is seroconverting to LV-like euPRRSV strains that the virus is responsible for problems. This diagnostic problem is one of the main reasons why most farmers did not vaccinate their piglets up till now in Western Europe. However, in pigs with flu-like problems caused by amPRRSV, subtype 2 and 3 euPRRSV and the new Flanders 13-like euPRRSV, it is easy to demonstrate the high replication of PRRSV in the upper respiratory tract by taking nasal swabs and blood and quantitating the high viral load (up to 105 TCID50/ml in nasal secretions and 104-6 TCID50/ml in blood) by virus titration or qRT-PCR.

PRRSV is a difficult target for the immunity. Several branches of the immunity have been shown not to be induced or not to be functional. Low levels of interferons are induced (Van Reeth et al., 1999). Antibodies are raised starting from 8 days post infection, but it takes several weeks before a weak neutralization can be demonstrated (Labarque et al., 2000). Natural killer cells and cytotoxic T-lymphocytes are not sufficiently effective (Cao et al., 2013; Costers et al., 2009). Only neutralizing antibodies, which appear after one month and at low levels together with a not yet identified porcine killer cell are the two branches that still can do the job and should be activated by vaccination. The drift of the virus makes it a moving target and complicates the whole vaccination strategy (Labarque et al, 2004). In the near future, it is important to have access to vaccines that are adaptable, enclosing strains that are closely related to the strains circulating in the field (Nauwynck et al., 2012). The technologies for making effective adaptable inactivated (Geldhof et al., 2012) and attenuated vaccines (unpublished data) became available and should be urgently implemented in the field. The ultimate dream is the development of an adaptable marker vector vaccine (cassette system) that induces a local immunity in the respiratory tract. As long as PRRSV does not persist such as its sister-arterivirus, lactate dehydrogenase elevating virus (LDV), we should keep on going with the investment to develop vaccines.

In conclusion, it is extremely important to better control PRRS in the near future and not to wait till a complete catastrophe occurs. There is an urgent need for adaptable inactivated and attenuated marker vaccines and improved biosafety measures in order to fully control PRRSV circulation. Pig producers, PRRS researchers and pharmaceutical companies should take their responsibility and join forces to come up with solutions to eradicate this ever-changing enemy that may turn into a real nightmare. In this context, funding PRRS research should be prioritized by all agencies all over the world. Not doing this is an unforgivable error not only for animal health but possibly also for human health.},
  author       = {Nauwynck, Hans and Frydas, Ilias and Christiaens, Isaura and Van Breedam, Wander and Bonckaert, Caroline and Trus, Ivan},
  booktitle    = {Proceedings of the 23rd International Pig Veterinary Society (IPVS) congress, vol. 1},
  keyword      = {viremia,immunity,PRRSV,PRRS,arterivirus,Nidovirales,subtype,Type},
  language     = {eng},
  location     = {Canc{\'u}n, Mexico},
  pages        = {23--26},
  publisher    = {International Pig Veterinary Society (IPVS)},
  title        = {Changing receptor use of PRRSV leads to different virological, immunological and clinical outcome: impact on diagnosis and control},
  url          = {http://www.ipvs2014.org/\_content/\_pdf/ProceedingsIPVS2014Volume-1.pdf},
  year         = {2014},
}