Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. METHODS: To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. RESULTS: Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. CONCLUSIONS: Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

Seroprevalence of Schmallenberg virus antibodies among dairy cattle, the Netherlands, winter 2011-2012.

Infections with Schmallenberg virus (SBV) are associated with congenital malformations in ruminants. Because reporting of suspected cases only could underestimate the true rate of infection, we conducted a seroprevalence study in the Netherlands to detect past exposure to SBV among dairy cattle. A total of 1,123 serum samples collected from cattle during November 2011-January 2012 were tested for antibodies against SBV by using a virus neutralization test; seroprevalence was 72.5%. Seroprevalence was significantly higher in the central-eastern part of the Netherlands than in the northern and southern regions (p<0.001). In addition, high (70%-100%) within-herd seroprevalence was observed in 2 SBV-infected dairy herds and 2 SBV-infected sheep herds. No significant differences were found in age-specific prevalence of antibodies against SBV, which is an indication that SBV is newly arrived in the country.

Development of a virus neutralisation test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds.

BACKGROUND: At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed. RESULTS: A virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. CONCLUSIONS: Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA's) and for (relative) quantification of antibodies.Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.

A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1.

BACKGROUND: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses.

Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection.

The level of heterosubtypic immunity (Het-I) and the immune mechanisms stimulated by a heterosubtypic influenza virus infection were investigated in pigs. Pigs are natural hosts for influenza virus and, like humans, they host both subtypes H1N1 and H3N2. Marked Het-I was observed when pigs were infected with H1N1 and subsequently challenged with H3N2. After challenge with H3N2, pigs infected earlier with H1N1 did not develop fever and showed reduced virus excretion compared with non-immune control pigs. In addition, virus transmission to unchallenged group-mates could be shown by virus isolation in the non-immune control group but not in the group infected previously with H1N1. Pigs infected previously with homologous H3N2 virus were protected completely. After challenge with H3N2, pigs infected previously with H1N1 showed a considerable increase in serum IgG titre to the conserved extracellular domain of M2 but not to the conserved nucleoprotein. These results suggest that antibodies against external conserved epitopes can have an important role in broad-spectrum immunity. After primary infection with both H1N1 and H3N2, a long-lived increase was observed in the percentage of CD8(+) T cells in the lungs and in the lymphoproliferation response in the blood. Upon challenge with H3N2, pigs infected previously with H1N1 again showed an increase in the percentage of CD8(+) T cells in the lungs, whereas pigs infected previously with H3N2 did not, suggesting that CD8(+) T cells also have a role in Het-I. To confer broad-spectrum immunity, future vaccines should induce antibodies and CD8(+) T cells against conserved antigens.

Vaccination of pigs with a DNA construct expressing an influenza virus M2-nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus.

In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.