Cold-adapted Virus Research Articles

Adaptation strategies of giant viruses to low-temperature marine ecosystems.

Microbes in marine ecosystems have evolved their gene content to thrive successfully in the cold. Although this process has been reasonably well studied in bacteria and selected eukaryotes, less is known about the impact of cold environments on the genomes of viruses that infect eukaryotes. Here, we analyzed cold adaptations in giant viruses (Nucleocytoviricota and Mirusviricota) from austral marine environments and compared them with their Arctic and temperate counterparts. We recovered giant virus metagenome-assembled genomes (98 Nucleocytoviricota and 12 Mirusviricota MAGs) from 61 newly sequenced metagenomes and metaviromes from sub-Antarctic Patagonian fjords and Antarctic seawater samples. When analyzing our data setalongside Antarctic and Arctic giant viruses MAGs already deposited in the Global Ocean Eukaryotic Viral database, we found that Antarctic and Arctic giant viruses predominantly inhabit sub-10°C environments, featuring a high proportion of unique phylotypes in each ecosystem. In contrast, giant viruses in Patagonian fjords were subject to broader temperature ranges and showed a lower degree of endemicity. However, despite differences in their distribution, giant viruses inhabiting low-temperature marine ecosystems evolved genomic cold-adaptation strategies that led to changes in genetic functions and amino acid frequencies that ultimately affect both gene content and protein structure. Such changes seem to be absent in their mesophilic counterparts. The uniqueness of these cold-adapted marine giant viruses may now be threatened by climate change, leading to a potential reduction in their biodiversity.

Development of reassortant influenza vaccines: classical reassortment or reverse genetics?

An important feature of influenza vaccines, which distinguishes them from other immunobiological preparations, is that they have no fixed composition. Due to the constant influenza virus antigenic variability, production facilities require timely supply with relevant vaccine strains undoable due to the lack of proper method for the convenient, rapid and uninterrupted development of vaccine strains. Among the licensed influenza vaccines, classical inactivated and live influenza vaccines hold a special place. They are based on reassortant vaccine strains obtained by crossing currently circulating influenza virus with the so-called donor strain (cold-adapted attenuation donor for live influenza vaccines or high yield donor for inactivated vaccines). Vaccine strains for licensed live attenuated influenza vaccines are reassortants with the so-called 6:2 genome formula two genes encoding hemagglutinin and neuraminidase (HA and NA) belong to the current epidemic virus, and six genes encoding internal proteins (PB2, PB1, PA, NP, M and NS) to cold-adapted master donor virus. There is a very limited number of donors of attenuation. In Russia, there are cold-adapted viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69; in the USA (MedImmune) there are viruses A/Ann Arbor/6/60ca (H2N2) and B/Ann Arbor/1/66ca. MedImmune produces vaccine strains using reverse genetics technique. For other countries, this approach for obtaining vaccines is limited due to the need to purchase a license from the patent holders. In Russia, genetic manipulations with strains for the seasonal live influenza vaccine are not yet allowed; reassortants for the Russian live influenza vaccine are created only by classical reassortment in embryonated chicken eggs. Vaccine candidates for the inactivated influenza vaccine are prepared by the classical reassortment method, the requirements for them are more flexible and allow to use diverse genes combinations from wild type virus and master donor virus. High-yielding viruses such as A/PR/8/34 (H1N1), A/Texas/1/77 (H3N2), B/Lee/40 and some others are used as donors of internal genes. Unfortunately, the classical reassortment method does not always allow to promptly obtain a reassortant virus with a 6:2 genome formula. This is hindered by a number of reasons, ranging from the unique properties of a certain epidemic virus ending up with the constellation of genes. The reverse genetics method based on plasmids is an alternative approach to create reassortant vaccine strains allowing to reliably and quickly obtain reassortant viruses of a set 6:2 genome formula. However, this method also has certain weaknesses. This review discusses the advantages and disadvantages of development of conventional influenza vaccine candidates by reverse genetics and classical reassortment in developing chick embryos.

Pantoea Bacteriophage vB_PagS_AAS23: A Singleton of the Genus Sauletekiovirus.

A cold-adapted siphovirus, vB_PagS_AAS23 (AAS23) was isolated in Lithuania using the Pantoea agglomerans strain AUR for the phage propagation. The double-stranded DNA genome of AAS23 (51,170 bp) contains 92 probable protein encoding genes, and no genes for tRNA. A comparative sequence analysis revealed that 25 of all AAS23 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. Based on the phylogenetic analysis, AAS23 has no close relationship to other viruses publicly available to date and represents a single species of the genus Sauletekiovirus within the family Drexlerviridae. The phage is able to form plaques in bacterial lawns even at 4 °C and demonstrates a depolymerase activity. Thus, the data presented in this study not only provides the information on Pantoea-infecting bacteriophages, but also offers novel insights into the diversity of cold-adapted viruses and their potential to be used as biocontrol agents.

Evaluation of influenza A and B cold-adapted reassortant virus reproduction in trivalent live influenza vaccines

BackgroundThe objective of the present study was to compare reproduction of trivalent LAIV vaccine strains in MDCK cells and to perform quantitative RT-PCR analysis of trivalent LAIV replication after inoculation in mice. MethodsWe applied a reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). We confirmed the PCR data in ELISA using staining of MDCK monolayer with mouse monoclonal antibodies to hemagglutinin. ResultsThe viral load during the reproduction of mono-vaccines and trivalent LAIV in MDCK cells was similar at low dilutions. The content of vaccine viruses was evaluated using quantitative RT-PCR analysis in the nasal turbinate and lungs of CBA mice on day 3 after intranasal immunization. It was shown that despite the almost complete absence of reproduction of the A/H3N2 virus in mice, the immune response of A/H3N2-specific antibodies was formed at the same level as to other viruses. In MDCK cells, a decreased infectious titers of vaccine viruses in trivalent LAIV compared to mono-vaccines was demonstrated except for B/Yamagata virus. ConclusionRT-PCR analysis is applicable to assess the growth characteristics of cold-adapted reassortant influenza viruses in vitro and in mice. The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. In mice, decrease in infectious titers did not lead to a decline of the immunogenicity.

Characterization of markers of cold-adapted candidate virus strains for live attenuated vaccines against chickenpox and shingles

Introduction. Chickenpox poses a significant public health concern due to its worldwide occurrence, a substantial probability of severe clinical progression, development of complications that can lead to a fatal outcome. Routine vaccination is the only way to prevent the disease. The purpose of this study was to assess the attenuation of cold-adapted (CA) candidate virus strains of Varicella zoster and Herpes zoster by using traditional and new methods.Materials and methods. The study was performed on strains of diploid cells from human embryonic lung and musculocutaneous tissue, primary and diploid cells of guinea pig fetal fibroblasts. Two clinical isolates of the virus were obtained — from a child with chickenpox and from an adult during the reactivation of shingles. The vOka vaccine strain and Ellen strain, a laboratory strain, were used as a control. The viral infectivity was measured by using a sensitive limiting dilution assay. The virulence was measured through the analysis of chick embryo chorioallantoic membranes infected with the Varicella zoster virus.Results. The clinical isolates were sub-cultured at lower temperatures, put through comparative tests and checked for presence of attenuation biomarkers. It was found that vFiraVax, a Varicella zoster virus strain, and vZelVax, a Herpes zoster virus strain were temperature-sensitive and cold-adaptable, but they lacked virulence. Attenuated CA virus strains induced lower expression of IFN-α and IFN-γ receptors on human mononuclear cells as compared to their parental variants.Conclusion. We created and assessed two candidate vaccine strains through attenuation of clinical isolates.

Nucleoprotein phosphorylation site (Y385) mutation confers temperature sensitivity to influenza A virus due to impaired nucleoprotein oligomerization at a lower temperature.

Mutations in viral proteins can lead to the cold adaption of influenza A virus and the cold-adapted virus is an important vaccination instrument. Here, we identify a novel strain of influenza A virus with cold sensitivity conferred by a mutation at a phosphorylation site within the nucleoprotein (NP). The highly conserved tyrosine 385 residue (Y385) of NP was identified as a phosphorylation site by mass spectrometry. The constructive NP phosphorylation mimicked by Y385E mutation was fatal for virus replication, while the continuous Y385 dephosphorylation mimicked by Y385F mutation had little impact on virus replication in vitro. Notably, the Y385F virus showed much lower replicative capacity in turbinates of mice compared with the wild type virus. Moreover, the replication of Y385F virus was significantly reduced in both A549 and MDCK cells grown at 33°C, when compared to that at 37°C. These results indicated that the Y385F mutation led to cold sensitivity of virus. We further found that the cold sensitivity of Y385F virus could be attributed to diminished NP oligomerization rather than any changes in intracellular localization. Taken together, these findings suggest that the phosphorylation of NP may be a critical factor that regulates the temperature sensitivity of influenza A virus.

Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.

Experimental Study of Genetic Constellation of Cold-Adapted Master Donor Viruses for Live Attenuated Influenza Vaccine Type B.

We studied the constellation of genes encoding polymerase complex proteins of master donor viruses for Russian live attenuated influenza vaccine type B. Reassortants of the reserve attenuation donor B/Leningrad/14/17/55 with B/USSR/60/69 master donor virus currently used for manufacturing seasonal influenza vaccine were prepared and examined. Most reassortants obtained by the classical reassortment method inherited all genes from the B/Leningrad/14/17/55 virus except the gene encoding PB1 subunit of the polymerase complex. One reassortant was selected for further evaluation of the role of PB1 gene. Greater attenuation of the strain for experimental animals (mice) in comparison with the original strains was demonstrated. This indicates high degree of constellation of genes of cold-adapted master donor viruses and the important compensating role of amino acid substitutions in the PB1 protein of B/Leningrad/14/17/55 donor virus in preventing viral hyperattenuation.

Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII).

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 °C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.

The Spatial Location of Single Amino Acid Substitutions in Proteins of Cold-Adapted Influenza B Viruses and Their Impact upon Cold Adaptation

This review summarizes the locations of unique amino acid substitutions in proteins of cold-adapted influenza B viruses, master donor viruses of live attenuated influenza vaccines: B/Ann Arbor/1/66ca, B/USSR/60/69, B/Leningrad/14/17/55, B/Victoria/2/63/87, and B/Vienna/1/99ca. The analysis is based on data on the viral sequences and information on the functional organization of internal proteins of the influenza B virus. A description is given of the main features of the structure of internal proteins from influenza B virus and a recent understanding of the functioning of their domains. We describe 35 substitutions in proteins of the ribonucleoprotein complex [PB1 (3), PB2 (10), PA (11), NP (11)] of cold-adapted influenza B viruses. In total, there are 44 amino acid positions that are considered to be associated with cold adaptation. Protein domains involved in protein–protein interactions are the most susceptible to changes. In the polymerase complex, the largest number of replacements were found in C-terminal domains of PA and PB2 proteins, forming the external parts of the polymerase complex structure and providing interaction among subunits, with cellular factors, and with the nucleoprotein (NP). Substitutions in the NP are located in the areas responsible for its oligomerization (flexible N-terminal fragment), as well as for interaction with the polymerase complex (the outer surface of the NP head and body domains). All the cold-adapted viruses had changes in the seventh segment of the genome, leading to amino acid substitutions in the matrix protein or BM2 proton channel. An analysis of the location of amino acid substitutions allows it to be theorized that an important role is played by the internal gene constellation in attenuation of cold-adapted viruses.

Development of Pyrosequencing-Based Assay for Analyzing the Origin of Genes in Preparing Reassortant LAIV Candidates

Live attenuated influenza vaccine (LAIV) is an effective measure to prevent influenza infection. The vaccine strain for LAIV is derived by reassortment of epidemic influenza strain with cold-adapted master donor virus (MDV) in developing chicken embryos. Molecular analysis of genomic composition of generated reassortants to select appropriate candidate for LAIV is a critical stage of vaccine strain development. Method of analysis permitting quick and precise detection of the genome composition of reassortants is required. The purpose of this study was to develop pyrosequencing-based assay using PyroMark Q24 for analysis of the origin of genes in preparing reassortant LAIV candidates for influenza A vaccine strains based on A/Leningrad/134/17/57 master donor virus and influenza B vaccine strains based on B/USSR/60/69 or B/Leningrad/14/77/55 master donor viruses, respectively. Objectives included development of primers set for genotyping of influenza A and B reassortants by pyrosequencing, establishing optimal dispensation order and assessment of effectiveness of protocol application for detecting the source of genes in virus samples. We have shown that a unified genome analysis protocol using PyroMark Q24 system is able to detect the source of internal genes in reassortant influenza strain by pyrosequencing. It allows analyzing the genome composition by a sensitive and specific innovative method in the first stages of vaccine strain development.

The M2 protein of live, attenuated influenza vaccine encodes a mutation that reduces replication in human nasal epithelial cells.

The influenza A virus components of the live, attenuated influenza vaccine (LAIV) encode the HA and NA gene segments from a circulating virus strain and the remaining gene segments from the cold-adapted master donor virus, A/Ann Arbor/6/1960 (H2N2). The master donor virus imparts at least three phenotypes: temperature-sensitivity (ts), attenuation (att), and cold-adaption (ca). The genetic loci responsible for the att and ts phenotypes of LAIV were mapped to PB1, PB2, and NP by reverse genetics experiments using immortalized cell lines. However, some in vivo studies have demonstrated that the M segment, which acquired an alanine (Ala) to serine (Ser) mutation at M2 position 86 during cold adaption - a mutation found in no other influenza A virus strain - contributes to the att phenotype. Prior studies have shown this region of the M2 cytoplasmic tail to be critical for influenza virus replication. Using reverse genetics, we demonstrate that certain amino acid substitutions at M2 positions 83 and 86 alter the replication of influenza A/Udorn/307/72 (H3N2). Importantly, substitution of a Ser at M2 position 86 reduces A/Udorn/307/72 replication in differentiated primary human nasal epithelial cell (hNECs) cultures, but does not considerably affect replication in MDCK cells. When a Ser was substituted for Ala at M2 86 in LAIV, the virus replicated to higher titers and with faster kinetics in hNEC cultures, implicating this amino acid change as contributing to LAIV attenuation. Increased replication also resulted in increased production of IFN-λ. These data indicate the LAIV associated Ser mutation at M2 position 86 contributes to the att phenotype and is associated with a differential regulation of interferon in LAIV infection.

H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets

Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virus-neutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model.

Phase I clinical trials of safety and immunogenicity of live cultural influenza vaccine vector-flu

Background Live influenza vaccines trigger all major components of the anti-flu immune response machinery and have been included in the global WHO program on the pandemic preparedness. Key advantages of using live vaccines include a feasibility of intranasal administration, opportunity to rapidly scale up production of the viral substance, simplicity of vaccination, and robust protection against antigenic drift variants of the virus. Use of certified cell cultures for the cultivation of seasonal strains of influenza along with biodegradable materials for constructing delivery vehicles is considered one of the mainstream approaches to the development of new generations of flu vaccines. We developed a live cultural influenza vaccine called Vector-Flu, which is based on the cold-adapted virus strain A/17/California/2009/38 (H1N1) and MDCK cell line obtained from the certified cell culture depository. Preclinical studies have demonstrated safety and high immunogenicity of Vector-Flu in a ferret model. Phase I of clinical trials was conducted on healthy volunteers in the Medical Unit #163 in Koltsovo, Russia. The trial pursued the following goals:1) Evaluation of safety and tolerability.2) Evaluation of the humoral and adaptive immune response using HI, ELISA and microneutralization assay.3) Evaluation of the cellular immune response, as measured by the cytokine release level in response to the ex vivo stimulation of blood lymphocytes by the influenza virus. Materials and methods Cell culture MDCK from Cell Culture Collection of SRC VB VECTOR. Cells were passed in serum-free SFM4MegaVir medium (USA). The characteristics of the MDCK cell line were studied in accordance with WHO [1].

A Single Dose of an Avian H3N8 Influenza Virus Vaccine Is Highly Immunogenic and Efficacious against a Recently Emerged Seal Influenza Virus in Mice and Ferrets.

H3N8 influenza viruses are a commonly found subtype in wild birds, usually causing mild or no disease in infected birds. However, they have crossed the species barrier and have been associated with outbreaks in dogs, pigs, donkeys, and seals and therefore pose a threat to humans. A live attenuated, cold-adapted (ca) H3N8 vaccine virus was generated by reverse genetics using the wild-type (wt) hemagglutinin (HA) and neuraminidase (NA) genes from the A/blue-winged teal/Texas/Sg-00079/2007 (H3N8) (tl/TX/079/07) wt virus and the six internal protein gene segments from the ca influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (H2N2), the backbone of the licensed seasonal live attenuated influenza vaccine. One dose of the tl/TX/079/07 ca vaccine induced a robust neutralizing antibody response against the homologous (tl/TX/079/07) and two heterologous influenza viruses, including the recently emerged A/harbor seal/New Hampshire/179629/2011 (H3N8) and A/northern pintail/Alaska/44228-129/2006 (H3N8) viruses, and conferred robust protection against the homologous and heterologous influenza viruses. We also analyzed human sera against the tl/TX/079/07 H3N8 avian influenza virus and observed low but detectable antibody reactivity in elderly subjects, suggesting that older H3N2 influenza viruses confer some cross-reactive antibody. The latter observation was confirmed in a ferret study. The safety, immunogenicity, and efficacy of the tl/TX/079/07 ca vaccine in mice and ferrets support further evaluation of this vaccine in humans for use in the event of transmission of an H3N8 avian influenza virus to humans. The human and ferret serology data suggest that a single dose of the vaccine may be sufficient in older subjects. Although natural infection of humans with an avian H3N8 influenza virus has not yet been reported, this influenza virus subtype has already crossed the species barrier and productively infected mammals. Pandemic preparedness is an important public health priority. Therefore, we generated a live attenuated avian H3N8 vaccine candidate and demonstrated that a single dose of the vaccine was highly immunogenic and protected mice and ferrets against homologous and heterologous H3N8 avian viruses.

African green monkeys recapitulate the clinical experience with replication of live attenuated pandemic influenza virus vaccine candidates.

Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. Importance: Ferrets and mice are commonly used for preclinical evaluation of influenza vaccines. However, we observed significant inconsistencies between observations in humans and in these animal models. We used African green monkeys (AGMs) as a nonhuman primate (NHP) model for a comprehensive and comparative evaluation of pairs of wild-type and pandemic live attenuated influenza virus vaccines (pLAIV) representing four subtypes of avian influenza viruses and found that pLAIVs replicate similarly in AGMs and humans and that AGMs can be useful for evaluation of the protective efficacy of pLAIV.

Characterization of reverse genetics-derived cold-adapted master donor virus A/Leningrad/134/17/57 (H2N2) and reassortants with H5N1 surface genes in a mouse model.

Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use.

Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate.

Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live Attenuated Influenza Vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuroaminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of vaccine candidates, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8 EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1 EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate.

Molecular Basis of Live-Attenuated Influenza Virus

Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.