Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05poly). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HAR65) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HATG05poly). In vitro, TG05poly and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05poly, TG05-HAR65, and R65-HATG05poly are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05poly led to temporary non-lethal disease in all animals, the reassortant TG05-HAR65 caused death in 3 of 10 animals. Furthermore, the reassortant R65-HATG05poly displayed the highest lethality as 8 of 10 chickens died, resembling “natural” HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins

Influenza B Virus With Modified Hemagglutinin Cleavage Site as a Novel Attenuated Live Vaccine

Background: Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component. Methods: As a novel attenuated live vaccine against influenza B virus, we generated a hemagglutinin cleavage site mutant of strain B/Lee/40 by replacing the common monobasic cleavage site recognized by trypsinlike proteases with an elastase-sensitive site, and we investigated the in vitro properties, attenuation, humoral responses, and efficacy in mice. Results. This mutant virus replicated in cell culture equally well as the wild type but in a strictly elastase-dependent manner. In contrast to the mouse-pathogenic parental virus, the cleavage site mutant was fully attenuated in mice and not detectable in their lungs. After 1 intranasal immunization, the animals survived lethal challenge with wild-type virus without weight loss or any other signs of disease. Furthermore, no challenge virus could be reisolated from the lungs of vaccinated mice. Conclusions: These findings demonstrate that proteolytic activation mutants can serve as live vaccine against influenza B virus

Avian Influenza Virus H3 Hemagglutinin May Enable High Fitness of Novel Human Virus Reassortants

<div><p>Reassortment of influenza A virus genes enables antigenic shift resulting in the emergence of pandemic viruses with novel hemagglutinins (HA) acquired from avian strains. Here, we investigated whether historic and contemporary avian strains with different replication capacity in human cells can donate their hemagglutinin to a pandemic human virus. We performed double-infections with two avian H3 strains as HA donors and a human acceptor strain, and determined gene compositions and replication of HA reassortants in mammalian cells. To enforce selection for the avian virus HA, we generated a strictly elastase-dependent HA cleavage site mutant from A/Hong Kong/1/68 (H3N2) (Hk68-Ela). This mutant was used for co-infections of human cells with A/Duck/Ukraine/1/63 (H3N8) (DkUkr63) or the more recent A/Mallard/Germany/Wv64-67/05 (H3N2) (MallGer05) in the absence of elastase but presence of trypsin. Among 21 plaques analyzed from each assay, we found 12 HA reassortants with DkUkr63 (4 genotypes) and 14 with MallGer05 (10 genotypes) that replicated in human cells comparable to the parental human virus. Although DkUkr63 replicated in mammalian cells at a reduced level compared to MallGer05 and Hk68, it transmitted its HA to the human virus, indicating that lower replication efficiency of an avian virus in a mammalian host may not constrain the emergence of viable HA reassortants. The finding that HA and HA/NA reassortants replicated efficiently like the human virus suggests that further HA adaptation remains a relevant barrier for emergence of novel HA reassortants.</p></div

Growth curves of MallGer05 HA reassortants.

<p>The gene segment composition of the each reassortant is given next to each chart: Hk68 genes (black) and MallGer05 genes (red). The presence of diverse additional MallGer05 segments is indicated by the abbreviation ‘div’. From supernatants of A549 cells infected with a MOI of 10⁻³, viral titers were determined by plaque assays on MDCK cells. The growth curves of the parental viruses Hk68 (black filled circles), Hk68-Ela (black empty circles) and MallGer05 (red filled circles) in each chart represent identical data and were drawn in for comparison. Growth curves were determined by plaque titrations of duplicate or quadruplicate infected cell cultures (in the latter case indicated by a diamond in the chart legend). Reassortants reaching titers at 48 h not less than one magnitude below Hk68 are highlighted by grey rectangles.</p

Growth curves of DkUkr63 HA reassortants.

<p>The gene segment constellation of each reassortant is given next to each chart: Hk68 genes (black) and DkUkr63 genes (red). The presence of diverse additional DkUkr63 segments is indicated by the abbreviation ‘div’. From supernatants of A549 cells infected with a MOI of 10⁻³, viral titers were determined by plaque assays on MDCK cells. The growth curves of the parental viruses Hk68 (black filled circles), Hk68-Ela (black empty circles) and DkUkr63 (red filled circles) in each chart represent identical data and were drawn in for comparison. Growth curves were determined by plaque titrations of duplicate or quadruplicate infected cell cultures (in the latter case indicated by a diamond in the chart legend). Reassortants reaching titers at 48 h not less than one magnitude below Hk68 are highlighted by grey rectangles.</p

Gene composition of DkUkr63 reassortants.

<p>Gene segments originating from DkUkr63 (D) and Hk68 (H) are shown in dark and light grey, respectively (HD indicates a mixed population). Co-infections with Hk68-Ela and DkUkr63 were performed either in the presence (+) or absence (−) of TPCK-treated trypsin. Supernatants were harvested either at eight or 24 hours after co-infection, the RNA isolated and subjected to RT-PCR for genotyping. Reassortants with no detectable HA titer in the supernatant after co-infection were propagated in embryonated chicken eggs and are indicated by a super-scripted ^e.</p

Influenza B Virus With Modified Hemagglutinin Cleavage Site as a Novel Attenuated Live Vaccine

Background: Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component. Methods: As a novel attenuated live vaccine against influenza B virus, we generated a hemagglutinin cleavage site mutant of strain B/Lee/40 by replacing the common monobasic cleavage site recognized by trypsinlike proteases with an elastase-sensitive site, and we investigated the in vitro properties, attenuation, humoral responses, and efficacy in mice. Results. This mutant virus replicated in cell culture equally well as the wild type but in a strictly elastase-dependent manner. In contrast to the mouse-pathogenic parental virus, the cleavage site mutant was fully attenuated in mice and not detectable in their lungs. After 1 intranasal immunization, the animals survived lethal challenge with wild-type virus without weight loss or any other signs of disease. Furthermore, no challenge virus could be reisolated from the lungs of vaccinated mice. Conclusions: These findings demonstrate that proteolytic activation mutants can serve as live vaccine against influenza B virus