Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.

BackgroundChikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.ResultsExpression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.ConclusionsChikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

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Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/ Kildare/89 at 10 6 50% egg infective doses (EID 50 )/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID 50 was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucleoprotein gene and a single set of primers from the matrix gene. RT-PCR using the matrix primers and virus isolation in embryonated eggs proved to be the most sensitive methods for the detection of virus. The Directigen Flu A test was the least sensitive method. The inclusion of 2% fetal calf serum in the viral transport medium inhibited the growth of virus from undiluted samples in MDCK cells but was essential for the maintenance of the virus titer in samples subjected to repeated freeze-thaw cycles.Equine influenza is considered to be the most important respiratory disease of the horse in the majority of countries where the breeding and racing of horses is a major industry. There are two subtypes of equine influenza virus, which is an orthomyxovirus: A/Equi 1/H7N7, first isolated in 1956 (20), and A/Equi 2/H3N8, first isolated in 1963 (24). Both subtypes have caused disease. However, it is generally accepted that A/Equi 1/H7N7 has not been isolated since 1979 and may be extinct (22,26). In contrast, A/Equi 2/H3N8 continues to circulate worldwide with the exception of a small number of island countries, such as Australia, New Zealand, and Iceland, where equine influenza has never been recorded. Infection with this subtype appears to be enzootic in North America and Europe, where two separate virus lineages have evolved and outbreaks frequently occur despite the mandatory vaccination of some horse populations (2, 10, 16a). However, it is immunologically naïve populations that are most at risk from equine influenza. Influenza is highly contagious, and the introduction of a single infected horse can result in explosive virus spread in unprotected horses over a wide geographical area. In South Africa in 1986, the introduction of the virus into the country for the first time resulted in thousands of horses suffering severe respiratory disease and necessitated the cancellation of racing for 5 months (5, 18).The risk associated with the increase in the international movement of horses by air transport for racing and breeding purposes means that it is imperative that there are sensitive virus detection systems available for the rapid diagnosis of equine influenza (21). Traditionally, equine influenza virus is diagnosed by the isolation of virus from nasopharyngeal swabs in embryonated hen eggs or by the detection of a fourfold-orgreater rise in antibody titer in paired sera by hemagglutination inhibition (16).The main objective of ...

A beta-glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both beta-glucosidase and beta-xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

BackgroundVirus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable.MethodsHere, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described.ResultsThe fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed.ConclusionsThe developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.