Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo.

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)-Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Variable impact of the hemagglutinin polybasic cleavage site on virulence and pathogenesis of avian influenza H7N7 virus in chickens, turkeys and ducks.

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.

A Dual Motif in the Hemagglutinin of H5N1 Goose/Guangdong-Like Highly Pathogenic Avian Influenza Virus Strains Is Conserved from Their Early Evolution and Increases both Membrane Fusion pH and Virulence.

Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by the acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required coadaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3,385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R and 124I but a frequency of 97.5% for the dual combination. This highly conserved dual motif is still retained in contemporary H5 HPAIV, including the novel H5NX reassortants carrying neuraminidases of different subtypes, like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/1997, carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S and124T, characteristic of low-pathogenic strains, prevented virus rescue, while the single substitutions attenuated the virus in both chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less-acidic endosomes, e.g., within the respiratory tract, resulting in an extended organ tropism. Taken together, HA coadaptation to increased acid sensitivity promoted the early evolution of H5 Goose/Guangdong-like HPAIV strains and is still required for their zoonotic potential.IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required coadaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic of low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less-acidic endosomes, e.g., within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted the emergence of H5 Goose/Guangdong-like HPAIV strains and is required for their zoonotic potential.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Multiple Introductions of Influenza A(H5N8) Virus into Poultry, Egypt, 2017.

After high mortality rates among commercial poultry were reported in Egypt in 2017, we genetically characterized 4 distinct influenza A(H5N8) viruses isolated from poultry. Full-genome analysis indicated separate introductions of H5N8 clade 2.3.4.4 reassortants from Europe and Asia into Egypt, which poses a serious threat for poultry and humans.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

BACKGROUND: Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. CASE PRESENTATION: Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. CONCLUSIONS: Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here 2.2.1.2a related to viruses in poultry in Israel, Gaza and Egypt. Enforcement of biosecurity and constant update of vaccine virus strains may be helpful to protect vaccinated birds and prevent spillover infection to neighbouring countries.

Biological fitness and natural selection of amantadine resistant variants of avian influenza H5N1 viruses.

Outbreaks caused by the highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry in several countries and posed a significant pandemic threat. In addition to culling of infected poultry and vaccination, amantadine has been applied in poultry in some countries to control the spread of the virus. The prevalence of the amantadine resistance marker at position 31 (Ser31Asn) of the M2 protein increased over time. However, little is known about the biological fitness and selection of H5N1 amantadine resistant strains over their sensitive counterparts. Here, using reverse genetics we investigated the biological impact of Ser31Asn in M2 commonly seen in viruses in clade 2.2.1.1 in farmed poultry in Egypt. Findings of the current study indicated that the resistance to amantadine conferred by Asn31 evolved rapidly after the application of amantadine in commercial poultry. Both the resistant and sensitive strains replicated at similar levels in avian cell culture. Asn31 increased virus entry into the cells and cell-to-cell spread and was genetically stable for several passages in cell culture. Moreover, upon co-infection of cell culture resistant strains dominated sensitive viruses even in the absence of selection by amantadine. Together, rapid emergence, stability and domination of amantadine-resistant variants over sensitive strains limit the efficacy of amantadine in poultry.

Composition of the Hemagglutinin Polybasic Proteolytic Cleavage Motif Mediates Variable Virulence of H7N7 Avian Influenza Viruses.

Acquisition of a polybasic cleavage site (pCS) in the hemagglutinin (HA) is a prerequisite for the shift of low pathogenic (LP) avian influenza virus (AIV) to the highly pathogenic (HP) form in chickens. Whereas presence of a pCS is required for high pathogenicity, less is known about the effect of composition of pCS on virulence of AIV particularly H7N7. Here, we investigated the virulence of four avian H7N7 viruses after insertion of different naturally occurring pCS from two HPAIV H7N7 (designated pCSGE and pCSUK) or from H7N1 (pCSIT). In vitro, the different pCS motifs modulated viral replication and the HA cleavability independent on the HA background. However, in vivo, the level of virulence conferred by the different pCS varied significantly. Within the respective viral backgrounds viruses with pCSIT and pCSGE were more virulent than those coding for pCSUK. The latter showed also the most restricted spread in inoculated birds. Besides the pCS, other gene segments modulated virulence of these H7N7 viruses. Together, the specific composition of the pCS significantly influences virulence of H7N7 viruses. Eurasian LPAIV H7N7 may shift to high pathogenicity after acquisition of "specific" pCS motifs and/or other gene segments from HPAIV.

Progressive glycosylation of the haemagglutinin of avian influenza H5N1 modulates virus replication, virulence and chicken-to-chicken transmission without significant impact on antigenic drift.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and continues to pose a pandemic threat. Studying the progressive genetic changes in A/H5N1 after long-term circulation in poultry may help us to better understand A/H5N1 biology in birds. A/H5N1 clade 2.2.1.1 antigenic drift viruses have been isolated from vaccinated commercial poultry in Egypt. They exhibit a peculiar stepwise accumulation of glycosylation sites (GS) in the haemagglutinin (HA) with viruses carrying, beyond the conserved 5 GS, additional GS at amino acid residues 72, 154, 236 and 273 resulting in 6, 7, 8 or 9 GS in the HA. Available information about the impact of glycosylation on virus fitness and pathobiology is mostly derived from mammalian models. Here, we generated recombinant viruses imitating the progressive acquisition of GS in HA and investigated their biological relevance in vitro and in vivo. Our in vitro results indicated that the accumulation of GS correlated with increased glycosylation, increased virus replication, neuraminidase activity, cell-to-cell spread and thermostability, however, strikingly, without significant impact on virus escape from neutralizing antibodies. In vivo, glycosylation modulated virus virulence, tissue tropism, replication and chicken-to-chicken transmission. Predominance in the field was towards viruses with hyperglycosylated HA. Together, progressive glycosylation of the HA may foster persistence of A/H5N1 by increasing replication, stability and bird-to-bird transmission without significant impact on antigenic drift.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens.

Highly pathogenic (HP) avian influenza viruses (AIV) evolve from low pathogenic (LP) precursors after circulation in poultry by reassortment and/or single mutations in different gene segments including that encoding NS1. The carboxyl terminal end (CTE) of NS1 exhibits deletions between amino acid 202 and 230 with still unknown impact on virulence of AIV in chickens. In this study, NS1 protein sequences of all AIV subtypes in birds from 1902 to 2015 were analyzed to study the prevalence and distribution of CTE truncation (ΔCTE). Thirteen different ΔCTE forms were observed in NS1 proteins from 11 HA and 8 NA subtypes with high prevalences in H9, H7, H6 and H10 and N9, N2, N6 and N1 subtypes particularly in chickens and minor poultry species. With 88% NS217 lacking amino acids 218-230 was the most common ΔCTE form followed by NS224 (3.6%). NS217 was found in 10 and 8 different HA and NA subtypes, respectively, whereas NS224 was detected exclusively in the Italian HPAIV H7N1 suggesting relevance for virulence. To test this assumption, 3 recombinant HPAIV H7N1 were constructed carrying wild-type HP NS1 (Hp-NS224), NS1 with extended CTE (Hp-NS230) or NS1 from LPAIV H7N1 (Hp-NSLp), and tested in-vitro and in-vivo. Extension of CTE in Hp NS1 significantly decreased virus replication in chicken embryo kidney cells. Truncation in the NS1 decreased the tropism of Hp-NS224 to the endothelium, central nervous system and respiratory tract epithelium without significant difference in virulence in chickens. This study described the variable forms of ΔCTE in NS1 and indicated that CTE is not an essential virulence determinant particularly for the Italian HPAIV H7N1 but may be a host-adaptation marker required for efficient virus replication.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens.

UNLABELLED: In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE: In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken.

Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant.

Chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived NDV immunity.

Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.

Identification and functional analysis of membrane proteins gD, gE, gI, and pUS9 of Infectious laryngotracheitis virus.

Herpesvirus envelope proteins are of particular interest for development of attenuated live, marker, and subunit vaccines, as well as development of diagnostic tools. The unique short genome region of the chicken pathogen infectious laryngotracheitis virus (ILTV, Gallid herpesvirus 1) contains a cluster of six conserved alphaherpesvirus genes encoding membrane proteins, of which up to now only glycoproteins gG and gJ have been analyzed in detail. We have now prepared monospecific rabbit antisera against ILTV gD, gE, and gI, and the ILTV type II membrane protein pUS9, each of which showed specific immunofluorescence reactions, and detected proteins of approximately 65 and 70 kDa (gD), 62 kDa (gI), 75 kDa (gE), or 37 kDa (pUS9) in western blot analyses of infected chicken cells. The proteins gD, gI, and gE, but not pUS9, were identified as abundant virion proteins, and gE and gI were shown to be N-glycosylated. We also isolated gE-, gI-, and pUS9-deleted ILTV recombinants, whereas it was not possible to purify gD-negative ILTV to homogeneity, indicating that gD, like in other alphaherpesviruses, is essential for receptor binding and virus entry. The pUS9-deleted ILTV exhibited almost wild-type-like replication properties in cell culture. The gE- and gI-negative viruses showed significantly reduced plaque sizes, whereas virus titers were barely affected. Since homologous gene-deletion mutants of other alphaherpesviruses are in use as live vaccines, the generated ILTV recombinants might be also suitable for this application.

Genetic changes that accompanied shifts of low pathogenic avian influenza viruses toward higher pathogenicity in poultry.

Avian influenza viruses (AIV) of H5 and H7 subtypes exhibit two different pathotypes in poultry: infection with low pathogenic (LP) strains results in minimal, if any, health disturbances, whereas highly pathogenic (HP) strains cause severe morbidity and mortality. LPAIV of H5 and H7 subtypes can spontaneously mutate into HPAIV. Ten outbreaks caused by HPAIV are known to have been preceded by circulation of a predecessor LPAIV in poultry. Three of them were caused by H5N2 subtype and seven involved H7 subtype in combination with N1, N3, or N7. Here, we review those outbreaks and summarize the genetic changes which resulted in the transformation of LPAIV to HPAIV under natural conditions. Mutations that were found directly in those outbreaks are more likely to be linked to virulence, pathogenesis, and early adaptation of AIV.

ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice.

Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5-ELP. ELPylated trimers could be purified by a membrane-based inverse transition cycling procedure with the potential of successful scale-up. The trimeric form of AIV HA was found to enhance the HA-specific immune response compared with the monomeric form. Plant-derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus-like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale-up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.