NP Gene Segments Research Articles

A novel reassortant clade 2.3.4.4 highly pathogenic avian influenza H5N6 virus identified in South Korea in 2018

Since 2017, clade 2.3.4.4b H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been detected over a broad geographic region, including Eurasia. These viruses have evolved through reassortment with Eurasian low pathogenic avian influenza viruses (LPAIVs), resulting in multiple genotypes. Here, we sequenced the full-length genome of 15 H5N6 HPAIVs collected from wild birds and poultry farms in South Korea from January to March 2018. A comparative phylogenetic analysis was then conducted. Three distinct genotypes were identified in South Korea during 2017/2018, including a novel reassortant genotype, H214. The novel reassortant H5N6 viruses isolated in this study possessed PB2, PA, and NP gene segments of Eurasian LPAIV on a genetic backbone of the H35-like genotype, which was identified in Korea and the Netherlands during 2017. Bayesian molecular clock analysis suggested that the novel reassortant viruses were generated most likely during the fall migration/wintering season of migratory waterfowl in 2017. Considering the continued emergence and spread of clade 2.3.4.4 HPAIV, enhanced surveillance of wild waterfowl is needed for early detection of HPAIV incursions.

Novel Highly Pathogenic Avian A(H5N2) and A(H5N8) Influenza Viruses of Clade 2.3.4.4 from North America Have Limited Capacity for Replication and Transmission in Mammals.

Highly pathogenic influenza A(H5N8) viruses from clade 2.3.4.4 were introduced to North America by migratory birds in the fall of 2014. Reassortment of A(H5N8) viruses with avian viruses of North American lineage resulted in the generation of novel A(H5N2) viruses with novel genotypes. Through sequencing of recent avian influenza viruses, we identified PB1 and NP gene segments very similar to those in the viruses isolated from North American waterfowl prior to the introduction of A(H5N8) to North America, highlighting these bird species in the origin of reassortant A(H5N2) viruses. While they were highly virulent and transmissible in poultry, we found A(H5N2) viruses to be low pathogenic in mice and ferrets, and replication was limited in both hosts compared with those of recent highly pathogenic avian influenza (HPAI) H5N1 viruses. Molecular characterization of the hemagglutinin protein from A(H5N2) viruses showed that the receptor binding preference, cleavage, and pH of activation were highly adapted for replication in avian species and similar to those of other 2.3.4.4 viruses. In addition, North American and Eurasian clade 2.3.4.4 H5NX viruses replicated to significantly lower titers in differentiated normal human bronchial epithelial cells than did seasonal human A(H1N1) and highly pathogenic A(H5N1) viruses isolated from a human case. Thus, despite their having a high impact on poultry, our findings suggest that the recently emerging North American A(H5N2) viruses are not expected to pose a substantial threat to humans and other mammals without further reassortment and/or adaptation and that reassortment with North American viruses has not had a major impact on viral phenotype. IMPORTANCE Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

Characterization of H9N2 influenza viruses isolated from Dongting Lake wetland in 2007

In 2007, a total of eight H9N2 influenza viruses were isolated from the water and fowl feces in Dongting Lake wetland, China. The genomes of the eight viruses were sequenced, and all eight gene segments were subjected to phylogenetic analysis. The results showed that all the isolates belonged to the same genotype, in which the HA, NA and NS gene segments were Chicken/Beijing/94-like; the PB2, PB1, PA and NP gene segments were Chicken/Shanghai/F/98-like; and the M gene was Quail/Hong Kong/G1/97-like. Animal experiments showed low pathogenicity of the selected viruses for chickens, although some chickens died after inoculation. The viruses showed no overt clinical signs in mice, but they could replicate in murine lungs prior to adaptation.

The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

We have previously determined that the temperature sensitive ( ts) and attenuated ( att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist ®), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 °C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 °C during a single-step replication, however, vRNA synthesis was reduced and the nuclear–cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 °C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.

Phenotypic properties resulting from directed gene segment reassortment between wild-type A/Sydney/5/97 influenza virus and the live attenuated vaccine strain

Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive ( ts) plaque formation and replication attenuation ( att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.

Genetic studies of FluMist™ influenza vaccines derived from cold-adapted A/Ann Arbor/6/60

The genetic basis for the attenuation ( att) and temperature sensitive ( ts) phenotypes of the cold-adapted A/Ann Arbor/6/60 influenza virus, the master donor virus for the influenza A (MDV-A) FluMist™ vaccine, was mapped to the PB1, PB2 and NP gene segments using plasmid-derived reassortants. The ts loci were introduced into A/Puerto Rico/8/34 and shown to confer both ts and att phenotypes to this divergent virus. This attenuated PR8 strain could potentially be used as a donor virus for manufacturing inactivated influenza vaccines against highly pathogenic strains.

Characterization of the attenuating M and NP gene segments of the avian influenza A/Mallard/78 virus during in vitro production of avian-human reassortant vaccine viruses and after replication in humans and primates

A unique requirement for live attenuated reassortant influenza vaccines is the need to generate new reassortant vaccine viruses with the appearance of each new antigenic variant. Thus, the attenuation phenotype conferred by the attenuated donor influenza virus must remain genetically stable during the generation of each new reassortant vaccine virus. In this study we used nucleotide sequence analysis to evaluate the genetic stability of the attenuating M and NP genes of the avian influenza A/Mallard/NY/6750/78 attenuated donor virus during the in vitro generation and subsequent in vivo replication of avian-human (AH) influenza A reassortant vaccine viruses in monkeys and humans. Nucleotide sequence changes in the M and NP genes occurred at a rate of ≈0.61 substitutions/1000 nt/reassortant during in vitro generation of four AH reassortant viruses. Only two nucleotide sequence changes occurred in the M and NP gene segments of four isolates of HINI or H3N2 AH vaccine viruses following 6–8 days of replication in seronegative children, and neither change affected amino acids previously identified as playing a potential role in attenuation. In addition, there were no changes in the nucleotide sequence of the M and NP genes of single gene AH reassortant viruses following five serial passages in squirrel monkeys. Finally, there was no change in the level or duration of replication of the single gene reassortant viruses in the upper or lower respiratory tract of monkeys following serial passage. These results suggest that nucleotide sequence changes occur infrequently during the production of reassortant influenza A vaccine viruses and their subsequent replication in humans and monkeys, and that genetic instability does not appear to represent a significant obstacle to this approach for production of live attenuated vaccines.

Regulation of the Replication of Influenza Virus RNA Segments: Partial Suppression of Protein Synthesis Restores the 'Early' Replication Pattern

The replication of influenza virus RNA was monitored by RNA-RNA hybridization with subsequent analysis of hybrid duplexes, as well as by immunosorption of viral nucleocapsids from extracts of [3H]uridine-labelled cells followed by the isolation and characterization of nucleocapsid-associated RNA. The nucleocapsid-associated RNA preparations contained mostly negative-strand genomic RNA. Electrophoresis of the hybrid RNA duplexes or single-stranded nucleocapsid-associated RNA in polyacrylamide gel revealed an 'early' replication pattern, with a predominance of the NP and NS gene segments, in cells labelled from 0 to 1 h post-infection. At later stages of infection the pattern changed to the 'late' one, with the M gene segment in excess of NS, and the NP gene no longer predominant. Cycloheximide added as late as 2 or 3 h post-infection suppressed RNA replication. Moderate concentrations of cycloheximide inhibited the replication of NS and NP gene segments to a lesser degree than the replication of the other RNA segments, thus restoring the 'early' replication pattern. Cycloheximide treatment resulted in a slight increase in the percentage of positive strands in nucleocapsid-associated RNA. The role of protein synthesis in the transition from the 'early' to the 'late' pattern of influenza virus RNA synthesis is discussed.