Polybasic Hemagglutinin Cleavage Site Research Articles

Genuine lethal infection of a wood pigeon (Columba palumbus) with high pathogenicity avian influenza H5N1, clade 2.3.4.4b, in Germany, 2022

Despite the increasing frequency of avian influenza (AI) cases in wild birds in Europe during the last decade, doves and pigeons were not recognized to be part of the AI epidemiology. Here we describe a natural, lethal HPAIV infection of subtype H5N1, clade 2.3.4.4b, in a wood pigeon (Columba palumbus) in Germany, 2022. The animal was housed in a bird sanctuary that suffered an HPAI-outbreak with multiple bird species affected. The pigeon’s post mortem findings were dominated by an acute lymphohistiocytic meningoencephalitis as well as neuronal necrosis in the grey matter of the cerebral hemispheres and in the brain stem. Influenza A viral antigen was associated to these alterations with a striking ependymal and periventricular distribution most probably indicating intraventicular liquorogenic spread of the virus. In addition, typical severe multifocal to coalescing necrotizing pancreatitis was evident. Occasionally, vascular endothelial cells showed an intense viral antigen staining. Examination of oropharyngeal and cloacal swabs and of various tissues by real-time RT-PCR corroborated systemic infection with highly pathogenic (HP) AIV of subtype H5N1. Viral loads soared in brain and pancreas samples. Full genome sequencing directly from brain tissue confirmed the assignment of the virus to clade 2.3.4.4b of the goose/Guangdong lineage bearing a polybasic hemagglutinin cleavage site. Our results demonstrate the principal susceptibility of wood pigeons to HPAIV H5N1 resulting in a sporadic infection. Considering the wide distribution and roaming of wild, feral, and racing pigeons with often close contact to humans, thorough investigation of suspected cases including pathological manifestation is advisable.

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

H7N9 Influenza Virus Containing a Polybasic HA Cleavage Site Requires Minimal Host Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.

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.

Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 106 EID50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1days) than that associated with Gifu/01 (2.5days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

H9N2 avian influenza virus-derived natural reassortant H5N2 virus in swan containing the hemagglutinin segment from Eurasian H5 avian influenza virus with an in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site

We isolated a novel H5N2 avian influenza virus from swans in China. The virus was derived from a widespread H9N2 avian influenza virus but acquired the hemagglutinin gene from Eurasian H5 subtype with a naturally occurring in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site.

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.

Amino acid substitution at position 44 of matrix protein 2 of an avirulent H5 avian influenza virus is crucial for acquiring the highly pathogenic phenotype in chickens

The pathogenicity of highly pathogenic avian influenza (HPAI) viruses is dependent on multiple factors, but the sequence at the HA cleavage site plays the most important role. To better understand the mechanism of virulence of HPAI virus, an avirulent H5 avian influenza virus, A/teal/Tottori/150/02 (H5N3, teal/150), was passaged in respiratory organs of chickens to generate a virus with a highly pathogenic phenotype. After 12 consecutive passages, the virus (strain 12a) became highly pathogenic, with a 100 % mortality rate in chickens. Sequence analysis of the highly pathogenic variant revealed an amino acid change from aspartic acid (Asp) to asparagine (Asn) at position 44 of matrix protein 2 (M2). To investigate the role of M2 in the pathogenicity of HPAI virus, we generated reassortant viruses possessing a polybasic HA cleavage site and either Asp or Asn at position 44 of M2 using the highly pathogenic strain 12a and the avirulent strain 7a, which has Asp at position 44 of M2 derived from isolate teal/150, and we compared their pathogenicity in chickens. Experimental infections demonstrated that the pathogenicity of viruses possessing Asp in M2 was dramatically decreased, and the mortality rate of inoculated chickens was 0 %, in contrast to viruses with Asn, which showed 70 to 100 % mortality. Our findings indicate that M2 protein of the avirulent H5 avian influenza virus is important for acquiring high virulence and that Asn at position 44 of M2, in addition to the polybasic HA cleavage site, is crucial for high pathogenicity in chickens.

The pathogenicity of swan derived H5N1 virus in birds and mammals and its gene analysis.

BackgroundHighly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in poultry and can infect and cause mortality in birds and mammals; the genetic determinants of their increased virulence are largely unknown. The main purpose of this work was to determine the correlation between known molecular determinants of virulence in different avian influenza virus (AIV) genes and the results of experimental infection of birds and mammals with AIV strain A/swan/Mangistau/3/06 (H5N1; SW/3/06).Methods and resultsWe examined the virulence of SW/3/06 in four species of birds (chickens, ducks, turkeys, geese) and five species of mammals (mice, guinea pigs, cats, dogs, pigs), and identified the molecular determinants of virulence in 11 genes (HA, NA, PB1, PB1-F2, PB2, PA, NS1, NS2, M1, M2 and NP). SW/3/06 does not possess the prime virulence determinant of HPAIV – a polybasic HA cleavage site – and is highly pathogenic in chickens. SW/3/06 replicated efficiently in chickens, ducks, turkeys, mice and dogs, causing 100% mortality within 1.6–5.2 days. In addition, no mortalities were observed in geese, guinea pigs, cats and pigs. The HI assay demonstrated all not diseased animals infected with the SW/3/06 virus had undergone seroconversion by 14, 21 and 28 dpi. Eleven mutations in the seven genes were present in SW/3/06. These mutations may play a role in the pathogenicity of this strain in chickens, ducks, turkeys, mice and dogs. Together or separately, mutations 228S-103S-318I in HA may play a role in the efficient replication of SW/3/06 in mammals (mice, dogs, pigs).ConclusionsThis study provides new information on the pathogenicity of the newly-isolated swan derived H5N1 virus in birds and mammals, and explored the role of molecular determinants of virulence in different genes; such studies may help to identify key virulence or adaptation markers that can be used for global surveillance of viruses threatening to emerge into the human population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0207-y) contains supplementary material, which is available to authorized users.

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins, referred to as "antigenic drift" and "antigenic shift." To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged 50 times in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum. The resulting mutant acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected, in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity.

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.

Acquisition of a Polybasic Hemagglutinin Cleavage Site by a Low-Pathogenic Avian Influenza Virus Is Not Sufficient for Immediate Transformation into a Highly Pathogenic Strain

Highly pathogenic avian influenza viruses (HPAIV) differ from all other strains by a polybasic cleavage site in their hemagglutinin. All these HPAIV share the H5 or H7 subtype. In order to investigate whether the acquisition of a polybasic cleavage site by an avirulent avian influenza virus strain with a hemagglutinin other than H5 or H7 is sufficient for immediate transformation into an HPAIV, we adapted the hemagglutinin cleavage site of A/Duck/Ukraine/1/1963 (H3N8) to that of the HPAIV A/Chicken/Italy/8/98 (H5N2), A/Chicken/HongKong/220/97 (H5N1), or A/Chicken/Germany/R28/03 (H7N7) and generated the recombinant wild-type and cleavage site mutants. In contrast to the wild type, multicycle replication of these mutants in tissue culture was demonstrated by positive plaque assays and viral multiplication in the absence of exogenous trypsin. Therefore, in vitro all cleavage site mutants resemble an HPAIV. However, in chicken they did not exhibit high pathogenicity, although they could be reisolated from cloacal swabs to some extent, indicating enhanced replication in vivo. These results demonstrate that beyond the polybasic hemagglutinin cleavage site, the virulence of HPAIV in chicken is based on additional pathogenicity determinants within the hemagglutinin itself or in the other viral proteins. Taken together, these observations support the notion that acquisition of a polybasic hemagglutinin cleavage site by an avirulent strain with a non-H5/H7 subtype is only one among several alterations necessary for evolution into an HPAIV.

The potential of a protease activation mutant of a highly pathogenic avian influenza virus for a pandemic live vaccine

The most effective countermeasure against a pandemic originating from a highly pathogenic avian influenza virus (HPAIV) is immunoprophylaxis of the human population. We present here a new approach for the development of a pandemic HPAIV live vaccine. Using reverse genetics, we replaced the polybasic hemagglutinin cleavage site of an H7N7 HPAIV with an elastase motif. This mutant was strictly elastase-dependent, grew equally well as the wild-type in cell culture and was attenuated in mice unlike the lethal wild-type. Immunization at 10(6)pfu dosage protected mice against disease and induced sterile immunity; vaccination with homosubtypic or heterosubtypic reassortants led to cross-protection. These observations demonstrate that a mutated hemagglutinin requiring elastase cleavage can serve as an attenuating component of a live vaccine against HPAIV.