Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2′-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens

Caractérisation de facteurs impliqués dans l'adaptation des virus influenza à leurs hôtes : exemple de la protéine NS1

Afin de mieux comprendre l'implication de la protéine NS1 dans la pathogénicité des virus influenza aviaires, nous avons reconstitué par génétique inverse la souche A/Turkey/Italy/977/1999 (H7N1). Dans un premier temps, nous avons produit un virus mutant exprimant une NS1 fortement tronquée, nommé H7N1 1-99. Ce virus, bien qu'incapable de se répliquer efficacement dans des fibroblastes embryonnaires de canard (DEF), conserve une capacité réplicative similaire à celle du virus sauvage lors de l'infection de cellules Vero, qui ne peuvent pas produire d'interférons de type I. En outre, le virus H7N1 1-99, à la différence du virus sauvage, a induit une forte production d'interférons de type I lors de l'infection de DEF. Dans un second temps, nous avons étudié les conséquences du polymorphisme C-terminal de la protéine NS1 lors de l'infection de diverses espèces. Pour cela, nous avons produit un virus exprimant une NS1 avec d'un motif C-terminal typiquement aviaire de séquence ESEV (nommé H7N1 ESEV) ainsi qu'un virus mutant dont la NS1 possède le motif RSKV (nommé H7N1 RSKV) préférentiellement associé aux souches humaines. Lors de l'infection de cellules humaines, le motif RSKV a conféré une plus grande capacité réplicative au virus. En revanche, sur cellules murines, le motif ESEV, aviaire, a rendu le virus plus pathogène. Enfin, Le virus H1N1 RSKV s'est répliqué plus efficacement lors de l'infection de canard tout en induisant une plus forte transcription de Mx, un gène induit par les interférons de type I. Ces données montrent que le domaine C-terminal de la protéine NS1 confère une pathogénicité qui dépend des espèces d'hôtes infectés.Avian influenza viruses represent a major public health concern. Particularly, the NS1 viral protein, which counteracts the innate immune response, is more and more considered as a virulence factor. How in order to understand how NS1 contributes to avian influenza pathogenicity, we used reverse genetics to reconstitute the avian strain A/Turkey/Italy/977/1999 (H7N1). We first generated a mutant virus expressing a truncated protein of 99 aa, named H7N1 1-99. This virus, whose replication is impaired in duck embryonic fibroblasts (DEF), remains able to replicate to high titres in the interferon deficient cell line Vero. Unlike the wild-type virus, the mutant virus induces a strong interferon production in DEF. These data underline the role of the NS1 protein during the infection of duck cells. In a second step, we evaluated the consequences of C-terminal polymorphisms of NS1 during the infection of several species. We therefore generated a virus whose NS1 protein carries the C-terminal avian sequence ESEV (named H7N1 ESEV) and a mutant virus expressing a NS1 protein with the “human-like” C-terminal sequence RSKV. Viral phenotypes were evaluated in vitro on human, murine or duck cells and in vivo on mice and ducks. In human cells, the H7N1 RSKV virus displayed an increased replicative capacity. Interestingly, the H7N1 ESEV virus was more pathogenic in mouse, with an increased pathogenicity, pulmonary replicationand type I interferon production. In ducks, the H7N1 RSKV virus replicated more efficiently and induced a stronger transcription of Mx, a gene induced by type I interferon. Collectively, these data show that the C-terminal domain of NS1 is a species-depdendent virulence motif

Caractérisation de facteurs impliqués dans l'adaptation des virus influenza à leurs hôtes (exemple de la protéine NS1)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Protection patterns in duck and chicken after homo- or hetero-subtypic reinfections with H5 and H7 low pathogenicity avian influenza viruses: a comparative study.

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza

Viral excretion in upper airways (left) or in lower digestive tract (middle) and antibody response (right) to LPAIV H5N3 in ducks (top) or in chickens (bottom).

<p>Viral load in oropharyngeal swabs (A, D) and cloacal swabs (B, E) was expressed as viral RNA copies per sample and compared between control (naïves) and challenged birds two days (for chickens, corresponding to the peak of infection in lung) or three days (for ducks, corresponding to the peak of infection in cloacum) after inoculation. All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Antibody titration (C, F) was expressed as inverse dilution of serum used for measurement by ELISA (vertical scores) or haemagglutination inhibition method (HI, draught-board).</p

Antibody response to LPAIV H7N2 in ducks (top) or in chickens (bottom).

<p>Antibody titers were measured by ELISA (left) or HI method (right) from serum collected at the indicated time in days (d) after primary infection (H7 groups, black bars) or heterologous secondary infection (H5H7 groups, white bars) and were expressed as the reciprocal of the dilution used for measurement as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105189#s2" target="_blank">Materials and Methods</a> section. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Antibody response to the H5N3 virus in ducks (top) or in chickens (bottom).

<p>Antibody titers were measured by ELISA (left) or HI method (right) from serum collected at the indicated time in days (d) after primary infection (H5 groups, black bars) or secondary infection (H5H5 groups, white bars) and were expressed as the reciprocal of the dilution of serum used for measurement as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105189#s2" target="_blank">Materials and Methods</a> section. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Viral excretion in upper airways (left) or in lower digestive tract (right) of LPAIV H5N3 in ducks (top) or in chickens (bottom).

<p>Birds were either primo-infected at six weeks of age (H5 groups) or re-infected at six weeks of age with the same virus as that used three weeks before (H5H5 groups). Viral titrations were measured at 2 days (d2, for chickens), 3 days (d3, for ducks) or 8 days p.i. (d8, for both bird species) and were expressed as viral RNA copies per sample and compared between primo-infected (H5 groups, black bars) and re-infected birds (H5H5 groups, white bars). All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Viral excretion in lower digestive tract of ducks (A) or in upper airways of chickens (B) of LPAIV H7N2.

<p>Birds were either primo-infected at six weeks of age (H7 groups) or re-infected at six weeks of age with H7N2 virus, three weeks after H5N3 first inoculation (H5H7 groups). Viral titrations were measured 2 days (d2, for chickens) in oropharyngeal swabs, 3 days (d3, for ducks) in cloacal swabs, or 8 days (d8, for both birds species in their respective swabs) and were expressed as viral RNA copies per sample and compared between primo-infected (H7 groups, black bars) and re-infected birds (H5H7 groups, white bars). All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p