Enhanced isolation of influenza viruses in qualified cells improves the probability of well-matched vaccines.

Influenza vaccines are utilised to combat seasonal and pandemic influenza. The key to influenza vaccination currently is the availability of candidate vaccine viruses (CVVs). Ideally, CVVs reflect the antigenic characteristics of the circulating virus, which may vary depending upon the isolation method. For traditional inactivated egg-based vaccines, CVVs are isolated in embryonated chicken eggs, while for cell-culture production, CVV's are isolated in either embryonated eggs or qualified cell lines. We compared isolation rates, growth characteristics, genetic stability and antigenicity of cell and egg CVV's derived from the same influenza-positive human clinical respiratory samples collected from 2008-2020. Influenza virus isolation rates in MDCK33016PF cells were twice that of eggs and mutations in the HA protein were common in egg CVVs but rare in cell CVVs. These results indicate that fully cell-based influenza vaccines will improve the choice, match and potentially the effectiveness, of seasonal influenza vaccines compared to egg-based vaccines.

Divergent Human-Origin Influenza Viruses Detected in Australian Swine Populations.

Global swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia from 2012 to 2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus data set comprising >40,000 sequences sampled globally revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including the H1N1/1977, H1N1/1995, H3N2/1968, and H3N2/2003, and the H1N1 2009 pandemic (H1N1pdm09) influenza A viruses, and a genotype that contained gene segments derived from the past three pandemics (1968, reemerged 1977, and 2009). Of the six human-derived gene lineages, only one, comprising two viruses isolated in Queensland during 2012, was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3 to 44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine from 2012 to 2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as antigenic archives of human influenza viruses, raising the risk of reemergence in humans when sufficient susceptible populations arise.IMPORTANCE We describe the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations from 2012 to 2016, showing that these viruses are distinct from each other and from those isolated from swine globally. Whole-genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated during various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere, indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza virus for extensive periods; we show direct evidence of a sustained transmission for at least 4 years between 2012 and 2016.

Evidence for the Introduction, Reassortment, and Persistence of Diverse Influenza A Viruses in Antarctica.

Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of the H11N2 subtype in Adélie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses in a snowy sheathbill during 2014, we isolated a novel H5N5 subtype virus from a chinstrap penguin during 2015. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a >99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting the continued prevalence of this virus in Antarctica over multiple years. However, phylogenetic analysis of the H5N5 virus showed that the genome segments were recently introduced to the continent, except for the NP gene, which was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes, with the majority of its genome segments derived from North American lineage viruses but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages in Antarctica over multiple years, the recent introduction of gene segments from diverse regions, and reassortment between different AIV lineages in Antarctica, which together significantly increase our understanding of AIV ecology in this fragile and pristine environment. IMPORTANCE: Analysis of avian influenza viruses (AIVs) detected in Antarctica reveals both the relatively recent introduction of an H5N5 AIV, predominantly of North American-like origin, and the persistence of an evolutionarily divergent H11 AIV. These data demonstrate that the flow of viruses from North America may be more common than initially thought and that, once introduced, these AIVs have the potential to be maintained within Antarctica. The future introduction of AIVs from North America into the Antarctic Peninsula is of particular concern given that highly pathogenic H5Nx viruses have recently been circulating among wild birds in parts of Canada and the Unites States following the movement of these viruses from Eurasia via migratory birds. The introduction of a highly pathogenic influenza virus in penguin colonies within Antarctica might have devastating consequences.

Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring.

Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue Q136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production.

Detection of evolutionarily distinct avian influenza a viruses in antarctica.

ABSTRACT Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment. IMPORTANCE Avian influenza viruses (AIVs) are typically maintained and spread by migratory birds, resulting in the existence of distinctly different viruses around the world. However, AIVs have not previously been detected in Antarctica. In this study, we characterized H11N2 viruses sampled from Adélie penguins from two geographically different sites in Antarctica and show that the segmented AIV genome diverged between 49 and 80 years ago from other AIVs, with several genes showing similarity and shared ancestry with H3N8 equine influenza viruses. This study provides the first insight into the ecology of AIVs in Antarctica and highlights the potential risk of an introduction of highly pathogenic AIVs into the continent.

Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses.

BACKGROUND: Influenza B viruses belong to two antigenically and genetically distinct lineages which co-circulate in varying proportions in many countries. OBJECTIVE: To develop simple, rapid, accurate and robust methods to detect and differentiate currently circulating B-lineage viruses in respiratory samples and virus isolates. STUDY DESIGN: Haemagglutinin (HA) gene sequences from more than 6300 influenza B strains were analysed to identify signature sequences that could be used to distinguish between B-lineages and sublineages. RESULTS: Pyrosequencing and a real time PCR assays were developed to detect the major B-lineages (B/Victoria/2/87 or B/Yamagata/16/88) and pyrosequencing for a unique mutation was used to further differentiate the B/Yamagata viruses into two currently co-circulating subgroups. More than 300 influenza virus-containing samples, including original specimens, cell and egg grown viruses, were tested with a 100% accuracy. Furthermore, when the same PCR primers were used in an rRT-PCR assay, the two lineages could be differentiated by their distinct ranges of melting temperature with an overall accuracy of 99% for 158 samples tested. CONCLUSIONS: These new pyrosequencing and rRT-PCR methods have the potential to aid the rapid identification of influenza B-lineages for surveillance purposes and to increase the available data for bi-annual selection of viruses for updating influenza vaccines.

Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses.

BACKGROUND: In April 2009, an A(H1N1) influenza virus of swine lineage was detected in humans in the USA, and in just over a month has infected over 10,000 people in more than 40 countries. OBJECTIVES: To determine the performance of the Binax Now, BD Directigen EZ, and the Quidel QuickVue influenza rapid point-of-care (POC) tests for the detection of the recently emerged swine lineage A(H1N1) virus. METHODS: Swine lineage A(H1N1) and human seasonal influenza strains were cultured and then diluted to specific infectivity titres. Viral dilutions were assayed by the rapid POC tests and by real-time RT-PCR. RESULTS: All three of the rapid POC tests successfully detected the swine lineage A(H1N1) viruses at levels between 10(3) and 10(5) TCID(50)/ml (tissue culture infectious dose(50)), with the BD Directigen test demonstrating marginally greater sensitivity than the other two tests. Viral infectivity and RNA load data for viruses at the detection limit of the rapid test kits, suggested that both the Quidel and the Binax tests were less sensitive for the detection of swine lineage A(H1N1) viruses than for human seasonal strains. In comparison the BD Directigen demonstrated similar sensitivity when detecting swine lineage A(H1N1) and human seasonal viruses. CONCLUSIONS: The three rapid POC tests all detected the emergent swine lineage A(H1N1) virus when it was present at high virus concentrations. Early diagnosis of infection can assist in the rapid treatment. However the tests are significantly less sensitive than PCR assays and as such, negative results should be verified by a laboratory test.

Vaccination against human influenza A/H3N2 virus prevents the induction of heterosubtypic immunity against lethal infection with avian influenza A/H5N1 virus.

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains. Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza. The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.

Practical considerations for high-throughput influenza A virus surveillance studies of wild birds by use of molecular diagnostic tests.

Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future.

Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells.

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b- and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b-CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b-CD8+ DCs presented to CD8 cells, and migratory CD11b-CD8- DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b-CD11chi DCs were depleted using either CD11c-diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.

Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds.

Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus-host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds.

Vaccination against highly pathogenic avian influenza H5N1 virus in zoos using an adjuvanted inactivated H5N2 vaccine.

Highly pathogenic avian influenza (HPAI) H5N1 virus infections have recently caused unprecedented morbidity and mortality in a wide range of avian species. European Commission directive 2005/744/EC allowed vaccination in zoos under strict conditions, while reducing confinement measures. Vaccination with a commercial H5N2 vaccine with vaccine doses adapted to mean body weight per species was safe, and proved immunogenic throughout the range of species tested, with some variations between and within taxonomic orders. After booster vaccination the overall homologous geometric mean titre (GMT) to the vaccine strain, measured in 334 birds, was 190 (95% CI: 152-236), and 80.5% of vaccinated birds developed a titre of >or=40. Titres to the HPAI H5N1 virus followed a similar trend, but were lower (GMT: 61 (95% CI: 49-76); 61%>or=40). The breadth of the immune response was further demonstrated by measuring antibody titres against prototype strains of four antigenic clades of currently circulating H5N1 viruses. These data indicate that vaccination should be regarded as a beneficial component of the preventive measures (including increased bio-security and monitoring) that can be undertaken in zoos to prevent an outbreak of and decrease environmental contamination by HPAI H5N1 virus, while alleviating confinement measures.

Mallards and highly pathogenic avian influenza ancestral viruses, northern Europe.

Outbreaks of highly pathogenic avian influenza (HPAI), which originate in poultry upon transmission of low pathogenic viruses from wild birds, have occurred relatively frequently in the last decade. During our ongoing surveillance studies in wild birds, we isolated several influenza A viruses of hemagglutinin subtype H5 and H7 that contain various neuraminidase subtypes. For each of the recorded H5 and H7 HPAI outbreaks in Europe since 1997, our collection contained closely related virus isolates recovered from wild birds, as determined by sequencing and phylogenetic analyses of the hemagglutinin gene and antigenic characterization of the hemagglutinin glycoprotein. The minor genetic and antigenic diversity between the viruses recovered from wild birds and those causing HPAI outbreaks indicates that influenza A virus surveillance studies in wild birds can help generate prototypic vaccine candidates and design and evaluate diagnostic tests, before outbreaks occur in animals and humans.

Protection of mice against lethal infection with highly pathogenic H7N7 influenza A virus by using a recombinant low-pathogenicity vaccine strain.

In 2003, an outbreak of highly pathogenic avian influenza occurred in The Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, approximately 1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.

Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR.

The potency of live attenuated virus vaccines is determined by counting or titrating viable viruses in cell cultures. These classical potency tests have the drawback that they are time consuming and laborious and show a high laboratory-to-laboratory variation. In the present study we describe the development and validation of a fast method to measure the potency of measles in trivalent measles, mumps and rubella (MMR) vaccines using quantitative real-time PCR (qPCR). Vero cells were infected with serial dilutions of a trivalent vaccine or a trivalent reference with known potency. Virus was allowed to replicate and subsequently replicated virus was quantitated by qPCR using the LightCycler technology. The virus titer in vaccine samples was estimated against reference preparations using parallel line analysis. In comparison to the plaque assay, the qPCR infectivity assay was faster and less laborious, while accuracy and intermediate precision were similar.