Influenza Virus Reassortment Occurs with High Frequency in the Absence of Segment Mismatch

Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 10⁶ PFU inocula, 46%–86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections.     

Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles

A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90–99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.     

Progress and Challenge in Computational Identification of Influenza Virus Reassortment

Virologica Sinica - Genomic reassortment is an important evolutionary mechanism for influenza viruses. In this process, the novel viruses acquire new characteristics by the exchange of the intact...     

Kinetics of Coinfection with Influenza A Virus and Streptococcus pneumoniae

Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly Streptococcus pneumoniae, but the precise processes involved are unclear. To address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two S. pneumoniae strains, type 2 D39 or type 3 A66.1. We determined that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.     

H5N1亚型禽流感病毒基因重排后毒力的变化

[目的]为了探讨高致病性禽流感病毒对水禽致病性差异的分子致病机理.[方法]我们对从野鸭分离到的H5N1亚型禽流感病毒的生物学特性进行鉴定,其中A/mallard/Huadong/Y/2003(Y)是对麻鸭无致病性病毒,而 A/mallard/Huadong/S/2005(S)是对麻鸭高致病性病毒.利用反向遗传技术构建一系列单个和多个基因组合替换基因重排病毒,并验证重排病毒在麻鸭上的致病力.[结果]研究表明,PB2, PB1, PA(3P), HA单基因以及3P基因组合替换的使S病毒对麻鸭的毒力完全致弱,但相应的基因替换后仅使Y病毒对麻鸭的毒力略有上升.两病毒的其它基因对毒力影响较小.[结论]H5N1亚型禽流感病毒对麻鸭的致病力受多基因调控,且这种调控作用在不同病毒骨架上的影响不一致,强毒受影响程度远比弱毒的大.     

Functional Match between Influenza Virus Hemagglutinin and Neuraminidase Is Restored after Gene Reassortment

Influenza virus A (FluA) reassortants with low-functional neuraminidase (NA) of subtype N1 and hemagglutinin (HA) of subtypes H2, H3, H4, and H13 display virion aggregation and accumulate to a lower titer because sialyl residues are not completely removed from virion components. Nonaggregating variants of FluA (H13N1) were shown to result from a mutation that reduces the HA affinity for sialyl substrates. Amino acid substitution K156E, which increases a negative charge at the edge of the receptor-binding pocket of HA large subunit (HA1), was revealed in two independent variants. This substitution was the only difference between HA1 of the original reassortant and one of its variants and, therefore, accounted for restoration of the functional match between HA and NA.     

Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.     

Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment

Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.