A reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal-protein genes acquired enhanced pig-to-pig transmission after serial passages in swine

Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2: pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/ Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2: pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2: pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2: pH1N1 (P0), H9N2: pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2: pH1N1 (P0) >= five days, 4/6 direct-contact animals shed the H9N2: pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs

Heterologous prime-boost vaccination with H3N2 influenza viruses of swine favors cross-clade antibody responses and protection

The emergence of multiple novel lineages of H1 and H3 influenza A viruses in swine has confounded control by inactivated vaccines. Because of substantial genetic and geographic heterogeneity among circulating swine influenza viruses, one vaccine strain per subtype cannot be efficacious against all of the current lineages. We have performed vaccination-challenge studies in pigs to examine whether priming and booster vaccinations with antigenically distinct H3N2 swine influenza viruses could broaden antibody responses and protection. We prepared monovalent whole inactivated, adjuvanted vaccines based on a European and a North American H3N2 swine influenza virus, which showed 81.5% aa homology in the HA1 region of the hemagglutinin and 83.4% in the neuraminidase. Our data show that (i) Priming with European and boosting with North American H3N2 swine influenza virus induces antibodies and protection against both vaccine strains, unlike prime-boost vaccination with a single virus or a single administration of bivalent vaccine. (ii) The heterologous prime-boost vaccination enhances hemagglutination inhibiting, virus neutralizing and neuraminidase inhibiting antibody responses against H3N2 viruses that are antigenically distinct from both vaccine strains. Antibody titers to the most divergent viruses were higher than after two administrations of bivalent vaccine. (iii) However, it does not induce antibodies to the conserved hemagglutinin stalk or to other hemagglutinin subtypes. We conclude that heterologous prime-boost vaccination might broaden protection to H3N2 swine influenza viruses and reduce the total amount of vaccine needed. This strategy holds potential for vaccination against influenza viruses from both humans and swine and for a better control of (reverse) zoonotic transmission of influenza viruses

Efficacy Studies of a Trivalent Vaccine Containing PCV-2a, PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Days of Age and 3 Weeks Later against Porcine Circovirus 2 (PCV-2) Infection

Four studies under preclinical and clinical conditions were performed to evaluate the efficacy of a new trivalent vaccine against Porcine circovirus 2 (PCV-2) infection. The product contained inactivated PCV-1/PCV-2a (cPCV-2a) and PCV-1/PCV-2b (cPCV-2b) chimeras, plus M. hyopneumoniae inactivated cell-free antigens, which was administered to piglets in a two-dose regime at 3 days of age and 3 weeks later. The overall results of preclinical and clinical studies show a significant reduction in PCV-2 viraemia and faecal excretion, and lower histopathological lymphoid lesions and PCV-2 immunohistochemistry scores in vaccinated pigs when compared to non-vaccinated ones. Furthermore, in field trial A, a statistically significant reduction in the incidence of PCV-2-subclinical infection, an increase in body weight from 16 weeks of age to slaughterhouse and an average daily weight gain over the whole period (from 3 days of age to slaughterhouse) was detected in the vaccinated group when compared to the non-vaccinated one. Circulation of PCV-2a in field trial A, and PCV-2b plus PCV-2d in field trial B was confirmed by virus sequencing. In conclusion, a double immunization with a cPCV-2a/cPCV-2b/M. hyopneumoniae vaccine was efficacious against PCV-2 infection by reducing the number of histopathological lymphoid lesions and PCV-2 detection in tissues, serum, and faeces, as well as reducing losses in productive parameters.info:eu-repo/semantics/publishedVersio

Efficacy Studies of a Trivalent Vaccine Containing PCV-2a, PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Days of Age and 3 Weeks Later against Porcine Circovirus 2 (PCV-2) Infection

Four studies under preclinical and clinical conditions were performed to evaluate the efficacy of a new trivalent vaccine against Porcine circovirus 2 (PCV-2) infection. The product contained inactivated PCV-1/PCV-2a (cPCV-2a) and PCV-1/PCV-2b (cPCV-2b) chimeras, plus M. hyopneumoniae inactivated cell-free antigens, which was administered to piglets in a two-dose regime at 3 days of age and 3 weeks later. The overall results of preclinical and clinical studies show a significant reduction in PCV-2 viraemia and faecal excretion, and lower histopathological lymphoid lesions and PCV-2 immunohistochemistry scores in vaccinated pigs when compared to non-vaccinated ones. Furthermore, in field trial A, a statistically significant reduction in the incidence of PCV-2-subclinical infection, an increase in body weight from 16 weeks of age to slaughterhouse and an average daily weight gain over the whole period (from 3 days of age to slaughterhouse) was detected in the vaccinated group when compared to the non-vaccinated one. Circulation of PCV-2a in field trial A, and PCV-2b plus PCV-2d in field trial B was confirmed by virus sequencing. In conclusion, a double immunization with a cPCV-2a/cPCV-2b/ M. hyopneumoniae vaccine was efficacious against PCV-2 infection by reducing the number of histopathological lymphoid lesions and PCV-2 detection in tissues, serum, and faeces, as well as reducing losses in productive parameters

Efficacy Studies against PCV-2 of a New Trivalent Vaccine including PCV-2a and PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Weeks of Age

This study aimed to evaluate the efficacy of a new trivalent vaccine containing inactivated Porcine Circovirus 1-2a and 1-2b chimeras and a Mycoplasma hyopneumoniae bacterin administered to pigs around 3 weeks of age. This trivalent vaccine has already been proved as efficacious in a splitdose regimen but has not been tested in a single-dose scenario. For this purpose, a total of four studies including two pre-clinical and two clinical studies were performed. Globally, a significant reduction in PCV-2 viraemia and faecal excretion was detected in vaccinated pigs compared to non-vaccinated animals, as well as lower histopathological lymphoid lesion plus PCV-2 immunohistochemistry scorings, and incidence of PCV-2-subclinical infection. Moreover, in field trial B, a significant increase in body weight and in average daily weight gain were detected in vaccinated animals compared to the non-vaccinated ones. Circulation of PCV-2b in field trial A and PCV-2a plus PCV-2d in field trial B was confirmed by virus sequencing. Hence, the efficacy of this new trivalent vaccine against a natural PCV-2a, PCV-2b or PCV-2d challenge was demonstrated in terms of reduction of histopathological lymphoid lesions and PCV-2 detection in tissues, serum and faeces, as well as improvement of production parameters.info:eu-repo/semantics/publishedVersio

A role for swine in the adaptation of H9N2 avian influenza viruses to humans?: lessons from serial passages and transmission studies in pigs

Avian H9N2 viruses are endemic in poultry in many Eurasian countries and sporadically caused dead-end infections in humans and swine. However, since the late 90s, H9N2 viruses from poultry have acquired human-like receptor specificity, increasing the potential for adaptation to mammals and the threat to public health. In experimental studies in pigs, reassortant H9N2 viruses containing internal genes from the 2009 pandemic H1N1 (pH1N1) virus showed increased replication efficiency and transmissibility. Nevertheless, it remains unknown whether H9N2 virus can adapt to pigs by further mutations in the hemagglutinin (HA) and/or other genes. To investigate this we performed ten, blind pig serial passages with A/Quail/Hong Kong/G1/97. Virus titers in nasal swabs and all respiratory tract (RT) tissues were examined in both pigs of each passage and selected samples were sequenced. Wild-type virus detected in low to moderate titers in 100% of nasal swabs and nasal mucosa samples, but only 50% of lower RT tissues. At the fourth passage virus replication increased with 100% of the RT samples being positive, and this was associated with a substitution in the amino acid 225 (H3 numbering) of the receptor binding site of the HA. However, virus replication decreased again from passage six on and the virus was lost at passage ten. We subsequently performed transmission studies to compare the wild-type virus and the fourth passage virus with an endemic swine influenza virus (SIV), the pH1N1. With pH1N1, all 6 direct contact (dc) pigs shed virus high amount of virus (mean area under the curve (AUC) 19), whereas the wild-type virus was shed by all dc pigs for a shorter time and in lower amounts (mean AUC 3). Though the fourth pig passage virus showed better transmission than the wild-type (mean AUC 6). Our results suggest that fully adaptation of an avian H9N2 virus to pigs is a complex process and may require a combination of adaptation strategies

Enhanced pig-to-pig transmission of a reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal genes by serial passaging in pigs

Avian influenza viruses of H9N2 subtype are endemic in poultry in Asia and the Middle East. Since 1998, they sporadically have caused dead-end infections in humans and swine. Along with avian H7 and H5 influenza viruses, H9N2 is in the top-3 of the world health organization’s list of pandemic influenza virus candidates. On the other hand, the 2009 pandemic H1N1 (pH1N1) virus originated from and has become endemic in swine. Its internal gene cassette has extensively reassorted with established swine influenza viruses all over the world. This pH1N1 internal gene cassette appears to be highly compatible with the surface protein genes of H9N2. This finding has raised concerns about the possible emergence of H9N2 reassortants with public health implications in swine. Moreover, such reassortant viruses showed increased replication and transmissibility in the mouse and ferret model of influenza. The few studies in pigs have also suggested improved fitness of the reassortants as compared to the parental H9N2 virus. We aimed to evaluate if a reassortant H9N2 virus could become better adapted to pigs by serial passaging. To investigate this we performed pig transmission experiments with four different viruses: a reassortant virus containing A/Quail/Hong Kong/G1/97 (H9N2) surface genes and A/California/04/09 (pH1N1) internal genes; another reassortant virus with the same gene constellation that had undergone seven passages in pigs and both parental viruses, H9N2 and pH1N1. Replication efficiency in the porcine lower respiratory tract was minimal for the reassortant H9N2 virus, enhanced for the wholly avian H9N2 virus and greatest for the pig-passaged H9N2 reassortant, with 0, 55 and 93% of the lower respiratory tract samples testing positive for the respective viruses. In pig transmission studies, pH1N1 was excreted in high amounts by all three directly inoculated pigs and six out of six direct contact pigs (mean area under the curve (AUC) for the direct contact pigs of 19,7). In contrast, none of the contact pigs shed high amounts of the parental H9N2 or the original H9N2 reassortant virus (means AUC 3,0 and 4,7, respectively), pointing to inefficient virus transmission. Remarkably, the pig-passaged reassortant was excreted in high titers by four out of six contact animals, with a mean AUC of 16,1. The genetic analysis of the reassortant viruses, to identify which mutations appeared during serial passaging allowing enhanced transmission, is still pending. Our findings confirm that avian H9N2 surface genes are compatible with pH1N1 internal gene cassette, that pigs could serve as suitable intermediate host for the adaptation of reassortant H9N2 virus to mammals, and thus pose a risk of becoming pandemic in humans

Could avian H9N2 influenza viruses become a threat to swine?: lessons from experimental studies in the pig

Introduction: It is a classical but unproven hypothesis that pigs can serve as intermediate hosts between birds and humans in the generation of novel pandemic influenza viruses. Yet the single pandemic virus of likely swine origin is the 2009 pandemic H1N1 virus (2009 pdm), a virus with genetic components of swine, human and avian origin (reassortant). The latter virus has become well adapted to pigs and humans and is now widespread in both species. Avian H9N2 viruses are endemic in Eurasian poultry and they are considered potential pandemic candidates because they cause sporadic dead-end infections in pigs and humans. However, H9N2 viruses are not adapted to mammals since they lack capacity to spread within the human or swine population. We and other researchers have previously examined whether H9N2 viruses could become adapted to pigs by two strategies: serial pig passages which are known to force viruses to mutate; and reassorment replacing H9N2 internal genes by those of a swine adapted influenza virus (SIV), the 2009 pdm. Both approaches slightly enhanced virus replication and transmission, but transmission was still inefficient when compared with SIV. Thus, we aimed to examine whether such reassortant H9N2 virus could become better adapted to swine after serial pig passages. Materials and Methods: We performed four pig transmission experiments with four different viruses: a non-passaged reassortant virus containing A/quail/Hong Kong/G1/97 (H9N2) surface genes and A/California/04/09 (2009 pdm) internal genes; another reassortant virus with the same genetic constellation but passaged seven times in pigs and both parental viruses, the avian H9N2 and the 2009 pdm SIV. In each experiment 3 animals were individually housed and intranasally inoculated with the selected virus. Two days later, 2 direct contact animals were co-housed with each inoculated pig. Nasal swabs were collected daily from all pigs for virus titration. Results: All inoculated pigs excreted medium to high amounts of virus during at least 5 days. The 2009 pdm was the single virus for which all 6 contact pigs shed high amounts of virus during 5 days. As expected, the parental H9N2 and the non-passaged reassortant H9N2 viruses were not efficiently shed by any contact pig. In contrast, the pig-passaged reassortant H9N2 virus was shed in high amounts by 4 of the 6 contact pigs. Conclusion: Our data suggest that serial passages induced mutations in the H9N2 reassortant virus, which improved its replication and transmission. Genetic analysis of this virus is still pending. We demonstrated that adaptation of avian H9N2 viruses to pigs is a complex multi-step process. H9N2 could pose a threat for pigs and humans if this process would occur in nature

Cross-lineage antibody responses and protection upon vaccination with inactivated swine influenza virus vaccines

Commercial, inactivated swine influenza virus (SIV) vaccines offer a solid protection against the homologous virus and its drift variants. However, they offer limited cross-protection against distinct H1 or H3 lineages. Indeed, vaccines based on European H3N2 SIVs do not protect against North American H3N2 SIVs and vice versa. Given the continuous increase in the number of novel SIV lineages, there is a need for SIV vaccines that offer a broader protection. According to recent studies in the mouse and ferret model, consecutive vaccinations with antigenically distinct influenza viruses of a given subtype may elicit a broader antibody response and protection. This has led us to investigate whether this approach may also work for SIV vaccines. Two monovalent vaccines were prepared: one vaccine contained the European H3N2 SIV sw/Gent/172/08 (Eu H3N2), the other vaccine contained the North American H3N2 SIV sw/Pennsylvania/62170-1/10 (US H3N2). These viruses show only 80,2% amino acid identity in their HA1. An oil-in-water adjuvant was added to UV-inactivated vaccine viruses. Pigs were allocated to 4 groups that were vaccinated twice with one month interval: a challenge control group, two homologous prime-boost vaccination groups (Eu H3N2 - Eu H3N2 and US H3N2 - US H3N2), and a heterologous prime-boost vaccination group (Eu H3N2 - US H3N2). Four weeks after the second vaccination they were challenged intranasally with either the European or North American H3N2 SIV. Serum antibody titers were determined in hemagglutination-inhibition, virus-neutralization and neuraminidase-inhibition assays. Peripheral blood mononuclear cells were isolated to quantify numbers of IFN-γ and IgG secreting cells by ELISPOT assays. All pigs were euthanized at 3 days post challenge and tissues of the respiratory tract were collected for virus titrations and (histo)pathology. Both homologous prime-boost vaccination groups had high serum antibody titers against the vaccine virus and a complete protection against replication of that virus in the lungs. However, antibody titers and protection against the other H3N2 SIV lineage were lacking. Heterologous prime-boost vaccination, in contrast, induced high serum antibody titers against both virus lineages, which correlated with an increased number of IgG secreting cells. A complete protection against virus replication in the lungs was observed in all pigs challenged with the Eu H3N2 and in 3 of 5 pigs challenged with US H3N2. Virus titrations of other tissues are still pending. Our data suggest that it may be possible to protect pigs against multiple lineages of H3N2 SIV using a heterologous prime-boost vaccination strategy