Comparative In Vitro and In Vivo Analysis of H1N1 and H1N2 Variant Influenza Viruses Isolated from Humans between 2011 and 2016.

Influenza A virus pandemics are rare events caused by novel viruses which have the ability to spread in susceptible human populations. With respect to H1 subtype viruses, swine H1N1 and H1N2 viruses occasionally cross the species barrier to cause human infection. Recently isolated from humans (termed variants), swine viruses were shown to display great genetic and antigenic diversity, hence posing considerable public health risk. Here, we utilized in vitro and in vivo approaches to provide characterization of H1 subtype variant viruses isolated since the 2009 pandemic and discuss the findings in context with previously studied H1 subtype human isolates. The variant viruses were well adapted to replicate in the human respiratory cell line Calu-3 and the respiratory tracts of mice and ferrets. However, with respect to hemagglutinin (HA) activation pH, the variant viruses had fusion pH thresholds closer to that of most classical swine and triple-reassortant H1 isolates rather than viruses that had adapted to humans. Consistent with previous observations for swine isolates, the tested variant viruses were capable of efficient transmission between cohoused ferrets but could transmit via respiratory droplets to differing degrees. Overall, this investigation demonstrates that swine H1 viruses that infected humans possess adaptations required for robust replication and, in some cases, efficient respiratory droplet transmission in a mammalian model and therefore need to be closely monitored for additional molecular changes that could facilitate transmission among humans. This work highlights the need for risk assessments of emerging H1 viruses as they continue to evolve and cause human infections.IMPORTANCE Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infections. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no preexisting immunity is an ongoing threat to public health. Recently isolated variant H1 subtype viruses were shown to display extensive genetic diversity and in many instances were antigenically distinct from seasonal vaccine strains. In this study, we provide characterization of representative H1N1v and H1N2v viruses isolated since the 2009 pandemic. Our results show that although recent variant H1 viruses possess some adaptation markers of concern, these viruses have not fully adapted to humans and require further adaptation to present a pandemic threat. This investigation highlights the need for close monitoring of emerging variant influenza viruses for molecular changes that could facilitate efficient transmission among humans.

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

In vitro characterization of influenza A virus attachment in the upper and lower respiratory tracts of pigs.

The binding of influenza A viruses to epithelial cells in the respiratory tract of mammals is a key step in the infection process. Therefore, direct assessment of virus-host cell interaction using virus histochemistry (VH) will enhance our understanding of the pathogenesis of these new viruses. For this study, the authors selected viruses that represented the 4 main genetic clusters of North American swine H1 (SwH1) viruses, along with A/California/04/2009 H1N1 and a vaccine strain for the positive controls, and the virus label, fluorescein isothiocyanate (FITC), for the negative control. A group of 5 viruses containing a 2-amino acid insertion adjacent to the binding site of the hemagglutinin protein and their presumed ancestral viruses were also examined for changes in binding patterns. Viruses were bound to formalin-fixed paraffin-embedded, 6-week-old (6w) and adult pig tissues. Qualitative VH scores per respiratory zone ranged from + to +++, with bronchioles having the highest and most consistent scores, regardless of animal age. For the 6w bronchioles, a quantitative VH score was calculated using digital images of 5 bronchioles per tissue section using image analysis software. Significant differences in attachment were found among the SwH1 viruses (P < .0001) and among the ancestral and insertion viruses (P < .0001). These results provide new insights on virus binding to porcine respiratory epithelial cells and the usefulness of morphometric scores. The results also highlight limitations of in vitro techniques, including VH for predicting virulence and host range.

Impact of anti-T-cell therapy in the immunogenicity of seasonal influenza vaccine in kidney transplant recipients.

BACKGROUND: The influence of anti-T-cell therapy in the immunogenicity of the influenza vaccine in kidney transplant recipients remains unclear. METHODS: During the 2010 to 2011 influenza season, we evaluated the immune response to the inactivated trivalent influenza vaccine in kidney transplant recipients having received Thymoglobulin or basiliximab as induction therapy. A hemagglutination inhibition assay was used to assess the immunogenicity of the vaccine. The primary outcome was geometric mean titers of hemagglutination inhibition after influenza vaccination. RESULTS: Sixty patients (Thymoglobulin n=22 and basiliximab n=38) were included. Patients in the Thymoglobulin group were older (P=0.16), showed higher creatinine levels (P=0.16) and had more frequently received a previous transplant (P=0.02). There were no significant differences in geometric mean titers for any of the three viral strains between groups (P=0.69 for H1N1, P=0.56 for H3N2, and P=0.7 for B strain). Seroconversion to at least one viral strain was seen in 15 (68%) of 22 patients in the Thymoglobulin group and 28 (73%) of 38 in the basiliximab group (P=0.77). In patients vaccinated during the first year after receiving anti-T-cell therapy (n=25), there was a trend toward lower vaccine responses in the Thymoglobulin group. Patients who received Thymoglobulin showed lower CD4(+) cell counts and lower levels of IgM, at an average of 16.2 months after transplantation. A multivariate analysis showed that only the absence of mycophenolate was associated with a better vaccine response (odds ratio=9.47; 95% confidence interval, 1.03-86.9; P=0.047). CONCLUSION: No significant differences were seen in immunogenicity of the influenza vaccine in kidney transplant recipients having received either Thymoglobulin or basiliximab.

Influenza B virus with modified hemagglutinin cleavage site as a novel attenuated live vaccine.

BACKGROUND: Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component. METHODS: As a novel attenuated live vaccine against influenza B virus, we generated a hemagglutinin cleavage site mutant of strain B/Lee/40 by replacing the common monobasic cleavage site recognized by trypsinlike proteases with an elastase-sensitive site, and we investigated the in vitro properties, attenuation, humoral responses, and efficacy in mice. RESULTS: This mutant virus replicated in cell culture equally well as the wild type but in a strictly elastase-dependent manner. In contrast to the mouse-pathogenic parental virus, the cleavage site mutant was fully attenuated in mice and not detectable in their lungs. After 1 intranasal immunization, the animals survived lethal challenge with wild-type virus without weight loss or any other signs of disease. Furthermore, no challenge virus could be reisolated from the lungs of vaccinated mice. CONCLUSIONS: These findings demonstrate that proteolytic activation mutants can serve as live vaccine against influenza B virus.

[Etiotropic therapy of influenza: lessons from the last pandemic].

Analysis of the experience gained during the last pandemic of 'swine' influenza A (H1N1) sw1 is presented with reference to clinical studies and etiotropic therapy. The mechanism of development of severe pneumonia as a result of mutations at the binding site of hemagglutinin receptor enhancing a2'-3'-sialoside specificity and pneumotropism of the virus is described. The data on the efficiency of Ingavirin, a new Russian antiviral for the treatment of influenza, are reported.

Vaccinia virus infections in martial arts gym, Maryland, USA, 2008.

Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym.

Isolation and identification of a non-haemagglutinating strain of rabbit hemorrhagic disease virus from China and sequence analysis for the VP60 Gene.

A variant strain of rabbit hemorrhagic disease virus, designated "whn-1", was isolated and identified in China. The virus lacked haemagglutinating activity at 25, 37 and 4 degrees C, respectively, and gave negative results in the HAT after two passages in experimentally infected rabbits, but gave positive results in Agar Diffusion Reaction (ADR) and Counter Immunoelectrophoresis (CIE). Using electron microscopy, negatively stained particles of the RHDV isolate showed that the virions was approximately 35 nm in diameter. The capsid protein VP60 gene of whn-1 strain was cloned into pMD18-T vector by RT-PCR assays and sequenced. The obtained VP60 gene sequence has been submitted to GenBank with the accession number: DQ069280. The whole VP60 gene of whn-1 was 1740 nt in size and encodes 579 aa. Alignment with other 16 strains of RHDV in the world, including such "RHDVa" strains as France 99-05, France-Reu-00, Germany-Triptis and ChinaTP, in addition to RCV and EBHSV, showed that the homology of RHDV strains were 90.0-98.0% for nucleotide sequence, 94.3-99.0% for amino acid sequence, respectively. The results indicated that the sequences of VP60 gene of different RHDV isolates, including non-haemagglutinating whn-1 strain and low-haemagglutinating Rainham strain, were relatively highly homologous, and the major variant amino acid were located within region C (301-328 aa) and region E(344-434 aa), which were specific to "RHDVa" strains. Moreover, the molecular characterisation of VP60 protein of RHDV whn-1 strain, such as Hydrophilicity plot, Flexible regions, Antigenic index, etc., were compared with reference RHDV strains of Spanish-AST/89, France-99-5 and UK-Rainham in this article. From the experiment, it's concluded that, the "whn-1" strain is probably an antigenic variant of "RHDVa", and the 3 amino acids of Phe (304), Ala (305), Ser (309), and 5 amino acids of Gly (359), Asn (365), Ala (369), Ala (370), Asn (386), located in P2 region in the VP60 protein, probably played an important role in the haemagglutination activity.

Baculovirus surface-displayed hemagglutinin of H5N1 influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity.

Baculovirus-mediated transductions of viral glycoprotein genes into mice were shown to elicit protective immune response in several reports. Here we reported that, under the control of ie1 promoter from white spot syndrome virus, hemagglutinin (HA) gene of H5N1 influenza virus could be efficiently expressed in both insect and mammalian cells mediated by recombinant baculovirus vectors. The HA protein, partially cleaved as in H5N1 virus, was significantly displayed on the surface of baculovirus and conferred the viral particle hemagglutination activity. Intramuscular injection of the purified HA-displaying baculovirus into mice stimulated the production of antibodies with Hemagglutination inhibition and neutralization activities against tested influenza virus. These data suggested an alternative way to apply baculovirus as an immunizing reagent against influenza virus infection.

Evidence against the acidification hypothesis in cystic fibrosis.

The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed a model system to selectively perturb the pH of a subset of acidified compartments in polarized cells and determined the effects on various protein trafficking steps. We then tested whether these effects were observed in cells lacking wild-type CFTR and whether reintroduction of CFTR affected trafficking in these cells. Our model system involves adenovirus-mediated expression of the influenza virus M2 protein, an acid-activated ion channel. M2 expression selectively slows traffic through the trans-Golgi network (TGN) and apical endocytic compartments in polarized Madin-Darby canine kidney (MDCK) cells. Expression of M2 or treatment with other pH perturbants also slowed protein traffic in the CF cell line CFPAC, suggesting that the TGN in this cell line is normally acidified. Expression of functional CFTR had no effect on traffic and failed to rescue the effect of M2. Our results argue against a role for CFTR in the regulation of organelle pH and protein trafficking in epithelial cells.

[Influenza virus surveillance in Tama, Tokyo--viral examinations in 1996/97, 1997/98, 1998/99, and 1999/2000 seasons].

During the 1996/97, 1997/98, 1998/99, and 1999/2000 seasons, isolations of influenza virus and/or confirmations of hemagglutinin gene of virus from the throat swab of patients in 9 medical institutions in Tama, Tokyo were carried out by RT-PCR and tissue culture with MDCK cells. In 1996/97 and 1997/98 seasons, type A (H3N2) was the predominant type of influenza virus, which was 85% in the 1996/97 and 97% in the 1997/98, but small-scale outbreaks with type B virus was also confirmed in the 1996/97 season (during the 8th and 10th week). On the other hand, in the other two seasons, 2 different kinds of types of virus were discovered, which was type A (H3N2) and type B in 1998/99 season, and H3N2 and H1N1 of type A virus in 1999/2000 season. In the 1998/99 season, type A (H3N2) prevailed in the first half, and type B gradually replaced type A (H3N2) after the 5th week in 1999, whereas 2 different kinds of type A in the 1999/2000 mixed during the outbreak. From the presented data, the difference of mode by age groups and year could be explained. Namely, most specimens and positive cases in 1996/97 and 1997/98 seasons were those from low age groups (fifteen years of age and below). In 1998/99 and 1999/2000 seasons, the number of specimens from high age groups (sixteen years of age or over) accounted for above 50%, and about 70% of type A (H3N2)-positive cases. Contrary, the frequency of type B virus in 1998/99 season and type A (H1N1) in 1999/2000 season was higher in the low age groups than the high ones.

Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico.

In October of 1993, there was decreased egg production and increased mortality among Mexican chickens, in association with serologic evidence of an H5N2 influenza virus. First isolated from chickens in May of 1994, after spreading widely in the country, the virus caused only a mild respiratory syndrome in specific pathogen-free chickens. Because eradication of the virus by destruction of infected birds posed major obstacles to the poultry industry in Mexico, we were able to conduct a "field experiment" to determine the fate of an avirulent virus after repeated cycles of replication in millions of chickens. By the end of 1994, the virus had mutated to contain a highly cleavable hemagglutinin (HA), but remained only mildly pathogenic in chickens. Within months, however, it had become lethal in poultry. Nucleotide sequence analysis of the HA cleavage site of the original avirulent strain revealed R-E-T-R, typical of avirulent viruses and unlike the K-K-K-R sequence characterizing viruses responsible for the 1983 outbreak in poultry in the United States. Both mildly and highly pathogenic isolates contained insertions and a substitution of basic residues in the HA connecting peptide, R-K-R-K-T-R, which made the HA highly cleavable in trypsin-free chicken embryo fibroblasts. Phylogenetic analysis of the HA of H5 avian influenza viruses, including the Mexican isolates, indicated that the epidemic virus had originated from the introduction of a single virus of the North American lineage into Mexican chickens. This sequence of events demonstrates, apparently for the first time, the stepwise acquisition of virulence by an avian influenza virus in nature.

Analysis of the hemagglutination activity domains of the Venezuelan equine encephalomyelitis and eastern equine encephalomyelitis viruses.

The hemagglutination (HA) domains of the Venezuelan equine encephalomyelitis (VEE) and the eastern equine encephalomyelitis (EEE) viruses providing the interaction of virions and red blood cells were studied with the use of a panel of 17 hemagglutination inhibition (HI) monoclonal antibodies (MAbs). A highly conserved domain (C domain) forming alphavirus-group-reactive MAbs was identified in the E2 protein of the VEE and EEE viruses. These MAbs inhibited HA of the western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya and Pixuna viruses. The involvement of amino acid residues 59 and 232 in the formation of the C region was demonstrated by sequencing the gene encoding the E2 protein of three escape variants of the VEE virus.