In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression.

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

Parrot bornavirus in naturally infected Brazilian captive parrots: Challenges in viral spread control.

Psittaciform orthobornaviruses are currently considered to be a major threat to the psittacine bird population worldwide. Parrot bornavirus (PaBV) was identified recently in Brazil and, since then, few studies have been conducted to understand the epidemiology of PaBV in captive psittacine birds. In the present study, natural infections by PaBV in South American parrots were investigated in two breeding facilities: commercial (A) and conservationist (B). Thirty-eight psittacine of 21 different species were presented for postmortem examination. Tissue samples were collected and investigated for the presence of PaBV-RNA using RT-PCR. In addition, clinical information about these birds was used when available. PaBV infection was detected in 73.7% of all birds investigated, indicating a wide dissemination of this virus in both facilities. From birds investigated in aviary A, 66.7% showed clinical signs, 100% had typical lesions of proventricular dilatation disease (PDD), 100% had mild to severe proventricular dilatation and 88.9% were PaBV-positive. In birds from aviary B, 27.6% showed clinical signs, 65.5% had typical lesions of PDD, 62% had mild to severe proventricular dilatation and 69% were PaBV-positive. Neurological disease was observed more frequently than gastrointestinal disease. Sequencing analysis of the matrix gene fragment revealed the occurrence of genotype 4 (PaBV-4) in both places. About 15.8% of birds in this study are threatened species. We discussed the difficulties and challenges for controlling viral spread in these aviaries and implications for South American psittacine conservation. These results emphasize the urgent need to develop a national regulatory and health standard for breeding psittacine birds in the country.

[Evolution of influenza A(H3N2) virus from 2007 to 2012].

The results of the genetic studies of influenza viruses make it possible to understand their evolution and recommendations for vaccine strains content. In this work, the data of complete sequence of the HA, NA, and M2-protein for 34 strains of influenza A(H3N2) virus circulating in Russia during 2007-2012 are presented. The influenza strains were isolated in Ivanovsky Institute of Virology, Moscow, and some collaborating Russian centers. The results of the phylogenetic analysis showed the differences among strains, which were observed during the analyzed period; the evolution had direction from A/Brisbane/10/2007 to A/Perth/16/2009 and A/ Victoria/208/2009. Hemagglutinin of the influenza A(H3N2) virus strains had differences between strains of last two seasons and strains circulating before, in the antigenic sites A, B, D, and, to a lesser extent, C, and E. In the neuraminidase gene the mutations responsible for the resistance to oseltamivir (E119V, R292K, N294S) and zanamivir (Q136K) were not found. All isolates carry the S31N mutation in the M2 gene responsible for resistance to amantadine.

Antiserum against the conserved nine amino acid N-terminal peptide of influenza A virus matrix protein 2 is not immunoprotective.

The recent emergence and rapid spread of the pandemic H1N1 swine influenza virus reminded us once again of the need for a universal influenza vaccine that can elicit heterosubtypic protection. Here, we show the superior immunogenicity and immunoprotective capacity of the full-length matrix protein 2 ectodomain (M2e) peptide coupled to keyhole limpet haemocyanin (KLH) compared with the N-terminal 9 aa residues of M2e (SP1). Immunization with M2e-KLH protected mice against a lethal challenge with influenza A virus and significantly reduced weight loss and lung virus titres. In addition, passive transfer of serum raised in rabbits against M2e-KLH protected mice against a lethal influenza virus challenge, whereas serum from rabbits immunized with SP1-KLH did not. Nevertheless, immunofluorescence staining revealed that rabbit serum raised against SP1-KLH bound specifically to infected Madin-Darby canine kidney cells. We conclude that the peptide SP1 contains an immunogenic epitope that is not sufficient for immunoprotection.

Development of a latex agglutination test for detecting antibodies against avian influenza virus based on matrix 1 protein expressed in vitro.

The matrix 1 (M1) gene, present in all subtypes of avian influenza virus (AIV), was cloned and expressed in Escherichia coli. Reactivity of the expressed protein was confirmed by western blot. Subsequently, the M1 gene expression product was purified and used as the antigen to develop a latex agglutination test (LAT) for detecting antibodies against these conventional subtypes of AIV including AIV H3, H5, H7, and H9 from chicken sera. The LAT is specific for AIV, and no cross-reaction was shown with chicken antisera against other avian viruses. Compared with the hemagglutination inhibition test, the corresponding specificity, sensitivity, and correlation were 95.7%, 88.7%, and 89.0%, respectively, in detecting 491 serum samples from vaccinated chickens.

Strategies and limitations in dendrimeric immunogen synthesis. The influenza virus M2e epitope as a case study.

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.

Origin of the 2009 Mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein.

Triple-reassortant swine influenza A (H1) viruses, containing genes from avian, human, and swine influenza viruses, emerged and became an outbreak among humans worldwide. Over a 1,000 cases were identified within the first month, chiefly in Mexico and the United States. Here, the phylogenetic analysis of haemagglutin (HA), neuraminidase (NA), and matrix protein (MP) was carried out. The analysis showed that the H1 of this reassortant originated from American pigs, while NA and MP were more likely from European pigs. All of the 2009 isolates appear homogeneous and cluster together, although they are distinct from classical human A (H1N1) viruses.

[Detection of C-terminal 30 bp-deletion mutation of latent membrane protein 1 of EBV in childhood lymphoma].

OBJECTIVE: To study C-terminal 30 bp-deletion mutation of latent membrane protein 1 of the virus from childhood lymphoma. METHODS: Nested-PCR was used to amplify C-terminal of EBV-LMP1 from childhood lymphoma and non-lymphoma associated to EBV, including Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL)and reactive hyperplasia of lymph node (RL). Sequence analysis was performed on the positive PCR product. RESULTS: LMP1 with 30 bp deletion in C-terminal was detected in 11/25 HL,3/8 NHL and 5/15 RL cases respectively. There were no significant differences among HL, NHL and RL (P = 0.793). Sequence analysis showed that LMP1 detected in this study belongs to the following three subgroups: B95.8, China1 and China2. CONCLUSION: LMP1 with 30 bp deletion in C-terminal widely existed in childhood HL,NHL and RL. There was no correlation between special types of LMP1 and the diseases. There were 3 LMP1 subgroups of EBV in children's lymphoma in the cases studied, including B95.8, China 1 and China 2.

[Full length nucleotide sequence analysis of L and M segments of Hantavirus Q32 strain gene].

BACKGROUND: To study the full length L and M sequence of Hantavirus Q32 strain gene and explore its molecular characters. METHODS: The L and M segment cDNA of Hantavirus Q32 strain was amplified by RT-PCR. The purified PCR products were sequenced directly or cloned into pGEM-T Vector and then sequenced. RESULTS: The L genome segment of Q32 virus was found to be 6533 nucleotides in length. One large open reading frame was found located at bases 38 to 6493. This was predicted to encode an L protein 2151 amino acids in length with a molecular mass of 2.46 x 10(5). The M genome segment was 3616 nucleotides in length. One open reading frame was located at bases 41 to 3488. This was predicted to encode an M protein 1135 amino acids with a molecular mass of 1.26 x 10(5). CONCLUSION: The nucleotides sequence of M and L segments of strain Q32 was similar to that of other Hantavirus M and L segments. Deduced amino acid sequences of glycoprotein and RNA polymerase revealed high homologue to other Hantavirus.

Analysis of the matrix protein gene sequence of the Asian lineage of peste-des-petits ruminants vaccine virus.

The M gene nucleotide sequence of an Indian peste-des-petits ruminants (PPRV) vaccine virus ("PPRV Sungri/96") belonging to Asian lineage was determined. The gene is 1476 nucleotides long with a single open reading frame (ORF). The nucleotide and predicted amino acid sequence was compared with the homologous region of the African Lineage Vaccine virus "PPRV/Nigeria/75/1". The nucleotide sequence of the "PPRV Sungri/96" was 86% identical to that of "PPRV/Nigeria/75/1", while a homology of 93% and 95% could be observed in the ORF and amino acids level, respectively. The M gene encodes a protein of 335 amino acids, with a predicted molecular weight (MW) of 37.8 kDa. The ORF is flanked by a 3' untranslated region of 436 nucleotides and a high level of sequence divergence (approximately 30%) could be observed in this region between the vaccine viruses of Asian and African lineages. A high degree of conservation of several amino acids of this protein observed previously was also confirmed in this study.

Increased positive selection pressure in persistent (SSPE) versus acute measles virus infections.

We compared the extent of positive selection acting on acute and persistent strains of measles virus (MV). Far stronger positive selection was found in the fusion (F) and haemagglutinin (H) genes from subacute sclerosing panencephalitis (SSPE) compared to acute MV cases. Most of the positively selected sites identified in these surface glycoprotein genes from SSPE cases correspond to structural, functional or antigenic areas, and could not be explained by the effects of cell passaging. The correlations between selected sites and functional studies of MV are discussed in detail with reference to the maintenance of persistent infection. No positive selection was found in the matrix (M) gene from acute cases of MV and the effects of including hypermutated SSPE M gene sequences in phylogenetic inference were also explored. Finally, using H gene data, we estimated the rate of molecular evolution for SSPE strains as 3.4 x 10(-4) substitutions/site/year, which is similar to previous estimates obtained for acute strains.

Evolutionary stasis of M1 gene of human influenza A viruses and the possibility of their subtyping by restriction analysis of M1 gene polymerase chain reaction product.

Nucleotide (nt) and amino acid (aa) sequences of the M1 protein in 36 human influenza A viruses were analyzed. The neighbor joining tree of the nt sequences revealed several lineages associated with past epidemics of human influenza. However, the tree of aa sequences revealed only few specific lineages. This discrepancy in phylogeny between nt and aa sequences indicates that the M1 protein of human influenza A virus nearly reached an evolutionary stasis. A simple subtyping method of human influenza A viruses by restriction fragment length polymorphism (RFLP) analysis of M1 gene polymerase chain reaction (PCR) products is discussed.

Sequences of the N and M genes of the sigma virus of Drosophila and evolutionary comparison.

The genome of the sigma rhabdovirus of Drosophila melanogaster consists of six genes in the order 3' N-2-3-4-G-L 5'. The nucleotide sequences of the N and of the fourth genes were determined from cDNA clones. Each gene contained a single long open reading frame encoding polypeptides with predicted MW of 50 and 25 kDa, respectively. Evidence that these genes encode the nucleocapsid N and the matrix M proteins were obtained using antibodies raised against recombinant proteins derived from the cloned genes and expressed in Escherichia coli. The M and N predicted amino acid sequences were compared with those of other rhabdoviruses. The M protein of sigma virus shared a similar domain arrangement to the other M proteins, but it showed very little sequence conservation. The N protein of sigma virus showed no significant homology with its counterpart in SYNV or IHNV and VSHV; it did, however, show sequence homology with the N of four vesiculoviruses and two lyssaviruses. The extent of amino acid identity suggests that sigma virus occupies an intermediate evolutionary position between these two genera. Some conserved motifs in the M and N proteins were deduced from the comparisons.