SH Genes Research Articles

Immunogenicity and Cross-Protection Efficacy of a Genotype F-Derived Attenuated Virus Vaccine Candidate against Mumps Virus in Mice.

Mumps virus (MuV) causes an acute contagious human disease characterized by swelling of the parotid glands. Despite the near elimination of mumps in many countries, the disease has recurred, even in vaccinated populations, especially adolescents. Immunization effectivity of the genotype A vaccine strain Jeryl Lynn (JL) is declining as genotype A is no longer predominant; therefore, a new vaccine strain and booster vaccine are required. We generated a cell culture-adapted MuV genotype F called F30 and evaluated its immunogenicity and cross-protective activity against diverse genotypes. F30 genome nucleotide sequence determination revealed changes in the NP, L, SH, and HN genes, leading to five amino acid changes compared to a minimally passaged stock (F10). F30 showed delayed growth, smaller plaque size in Vero cells, and lower neurotoxicity in neonatal mice than F10. Furthermore, F30 was immunogenic to other genotypes, including the JL vaccine strain, with higher efficacy than that of JL for homologous and heterologous immunization. Further, F30 exhibited cross-protective immunity against MuV genotypes F and G in Ifnar-/- mice after a third immunization with F30 following two doses of JL. Our data suggest that the live-attenuated virus F30 could be an effective booster vaccine to control breakthrough infections and mumps epidemics.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNβ, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes

The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker’s yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.

In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Molecular epidemiology of human respiratory syncytial virus and human metapneumovirus in hospitalized children with acute respiratory infections in Croatia, 2014–2017

Acute respiratory infection (ARI) is the most common infection in children under 5 years of age and it is frequently caused by two pneumoviruses, human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). Epidemic seasons of these viruses overlap and disease manifestations are highly similar, including severe lower ARI such as bronchiolitis or pneumonia. Reinfections with pneumoviruses are frequent and limited prevention treatment is available. Genetic diversity of HRSV and HMPV strains circulating in Croatia was monitored during four consecutive years (2014–2017). Co-circulation of multiple lineages was observed for both viruses. Within HRSV group A, ON1 strains gained strong predominance during the 4-year period, while previously dominant genotype NA1 was detected only sporadically. Similarly, newly occurring HMPV genotype A2c gained predominance over genotype A2b during this period, resulting in all infection in 2017 being caused by A2c. Along with phylogenetic analysis based on the commonly used fragments for detection and genotyping of these viruses, full length G and SH genes were also analysed. Evolutionary dynamics showed that inferred substitution rates of HRSV and HMPV are between 2.51 × 10−3 and 3.61 × 10−3 substitutions/site/year. This study established presence of recently described HMPV strains containing large duplications in the G gene in Croatia. Viruses with either of the two duplications belong to a subcluster A2c, which has completely replaced all other group A subclusters in 2017.

Nucleotide and Amino Acid Changes in HN, F and SH genes of an Iranian Mumps Virus; RS-12, Following Attenuation to Vaccine Strain

Nucleotide and Amino Acid Changes in HN, F and SH genes of an Iranian Mumps Virus; RS-12, Following Attenuation to Vaccine Strain

Charged amino acids in the AMPV fusion protein have more influence on induced protection than deletion of the SH or G genes

Modifications to F, G and SH genes of an avian metapneumovirus (AMPV) field isolate were made by reverse genetics and their virulence and protective capacity were tested in young turkeys. Infection of one-day-old turkeys with a subtype A AMPV neither caused disease nor stimulated detectable protection against subsequent virulent challenge. While serial passage of this virus in tracheal tissue increased virulence, protection stimulated remained moderate. Substitution of the fusion protein from a protective AMPV very minimally increasing virulence but dramatically increased induced protection; and this was associated with five amino acid substitutions all involving charged amino acids which computational analysis predicted to affect protein surface properties but not immunodominant helper T-lymphocyte antigenic sites. When SH or G genes were deleted, viruses caused no disease but still conferred full protection to the majority of turkeys. In the case of the SH deletion, shed virus post-inoculation was undetectable. Partial SH deletions were found to confer protection related to the length of SH open reading frame remaining. Removal of both SH and G genes together produced a virus conferring negligible protection. We conclude that the characteristics of the AMPV fusion protein are important in inducing protection while the SH and G genes under investigation played a lesser role.

Seleção para a resistência à ferrugem em progênies das cultivares de café IPR 99 e IPR 107

As cultivares de café IPR 99 ("Sarchimor") e IPR 107 ('IAPAR 59' x 'Mundo Novo IAC 376-4') eram no passado resistentes à ferrugem (Hemileia vastatrix). Os objetivos deste estudo foram: a) Identificar progênies dessas duas cultivares com resistência à ferrugem; b) Identificar progênies com resistência incompleta; c) Verificar a eficiência dos cruzamentos testes na seleção dessas cultivares. A avaliação da resistência em campo foi realizada em cafeeiros adultos expostos à população local de raças presentes no IAPAR. Foram avaliados 23 cruzamentos testes com progênies F3 de 'IPR 107' e 5 com progênies F4 de 'IPR 99'. Além disso, foram avaliadas 11 progênies F4 de 'IPR 107' e 5 progênies F5 de 'IPR 99', provenientes de autofecundação. Várias progênies das cultivares IPR 99 e 107 apresentaram alta freqüência de plantas com resistência completa e são portadoras de mais genes de resistência não quebrados pelas raças de ferrugem. Progênies das cultivares IPR 99 e IPR 107 com a resistência quebrada apresentaram resistência incompleta à população local de raças. Cruzamentos testes foram eficientes na seleção de progênies de café com mais genes de resistência não quebrados.

Increased susceptibility of human respiratory syncytial virus to neutralization by anti‐fusion protein antibodies on adaptation to replication in cell culture

Subgroup A respiratory syncytial viruses present in respiratory secretions and low passage level cell culture isolates were found to be markedly less susceptible to neutralization with monoclonal antibodies (MAbs) to the F glycoprotein than the cell culture adapted A2 virus strain. Low passage virus isolates collected over a 20 year period and belonging to several sub-group A lineages were refractory to neutralization with antibodies recognizing two major neutralizing antigenic sites located sub-terminally at opposite ends of the F(1) glycoprotein sub-unit. On further passage in cell culture, virus isolates exhibited both increased infectivity titers and increased susceptibility to neutralization by antibodies to both antigenic sites. The consensus nucleotide sequence of the membrane associated proteins M and of the SH, G and F glycoprotein genes, and their intergenic regions were compared for neutralization resistant and susceptible stocks of one virus strain, R17532. No changes were observed in the known monoclonal antibody epitopes on the F glycoprotein. In line with this, the increase in susceptibility was not found to be associated with any increased binding of monoclonal antibody to isolated F glycoprotein in a BIAcore assay, thus excluding the possibility that passage in cell culture selected for viruses with mutations in the antibody binding sites. M and SH genes were conserved but a number of sites in the G and F glycoprotein genes were found to vary on adaptation to cell culture suggesting that change in susceptibility to neutralization was associated with a change in the prevalent quasispecies present in the virus population. The genetic basis of phenotypic change in susceptibility remains to be determined.

Genetic Variability of Human Metapneumovirus Infection: Evidence of a Shift in Viral Genotype without a Change in Illness

Human metapneumovirus (hMPV) was identified in 2001 as a cause of acute respiratory illness, but its characteristics are still being defined. We analyzed 3740 nasopharyngeal-wash specimens obtained during 2002-2004, using assays for common respiratory viruses and real-time polymerase chain reaction for hMPV. We detected hMPV in 5% of all specimens, compared with 28% for other respiratory viruses. Nucleotide sequence analysis of hMPV isolates revealed the predominant circulation of hMPV genotype A in the 2003 season but a switch to predominantly genotype B in 2004. Sequence analysis also revealed major differences in the hMPV G and SH genes but relative conservation of the F and N genes within each genotype. Phylogenetic analysis indicated a seasonal switch within hMPV genotype A subtypes as well. Despite genetic variability, we found no difference in the severity of illness caused by various hMPV isolates. These findings suggest that hMPV may vary in genetic structure, to allow for a seasonal shift in predominant genotype and the maintenance of infection rates.

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3′-N-P/V/C-M-F-SH-TM-G-L-5′. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3′ half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.

Evaluation of recombinant respiratory syncytial virus gene deletion mutants in African green monkeys for their potential as live attenuated vaccine candidates

Towards the goal of developing live attenuated respiratory syncytial virus (RSV) vaccines to prevent severe respiratory tract infections caused by respiratory syncytial virus, recombinant RSV containing a deletion of single or multiple NS1, NS2, SH and M2-2 genes have been generated. In this study, recombinants, rA2ΔM2-2, rA2ΔNS2, rA2ΔNS1NS2, rA2ΔSHNS2, rA2ΔM2-2NS2 were evaluated in African green monkeys (AGMs) for their infectivity, immunogenicity and protection against wild type (wt) RSV challenge. Replication of rA2ΔNS2 and rA2ΔSHNS2 was not attenuated in either the upper or the lower respiratory tracts of AGMs. On the other hands, rA2ΔNS1NS2 was over-attenuated; it did not replicate in the respiratory tracts of the infected monkeys and did not provide sufficient protection against wild type RSV challenge. rA2ΔM2-2NS2 was slightly more attenuated than rA2ΔM2-2 and provided partial protection against wt RSV challenge. rA2ΔM2-2, and possibly rA2ΔM2-2NS2, exhibited the attenuated but protective phenotypes in the monkeys that could be further evaluated as potential live attenuated RSV vaccine candidates in the clinical studies.

The failure of interleukin-10-deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen-sensitized/challenged mice.

Interleukin-10-deficient mice develop a robust pulmonary inflammatory response but no airway hyperresponsiveness (AHR) to inhaled methacholine (MCh) following allergen sensitization and challenge. In the present study, we investigated the effect of respiratory syncytial virus (RSV) infection on AHR and pulmonary inflammation in allergic IL-10-/- mice. Unlike littermate control mice, RSV-infected or ovalbumin (OVA)-sensitized/challenged IL-10-/- mice failed to develop significant AHR. In contrast, sensitized/challenged IL-10-/- mice infected with RSV did develop AHR accompanied by increased eosinophil numbers, both in bronchoalveolar lavage (BAL) and pulmonary tissue, and mucin production in airway epithelium. The cytokine profile in OVA-sensitized/challenged IL-10-/- mice was skewed toward a Th1 response but after RSV infection, this response was more of a Th2 type, with increased IL-5 levels in the BAL. Studies with an RSV mutant that lacks the G and SH genes showed equal enhancement of the AHR response as the parental wild-type strain, indicating that G protein is not essential to this response. These data suggest that RSV infection can overcome the failure of development of AHR in allergic IL-10-/- mice.

Revolutionary biology: the new, gene-centered view of life

There is a revolution underway in biology. It is based on a new perception of bodies and in which the former are the end product of the latter within the continuum of evolution. Twenty fi ve years after Richard Dawkins helped revolutionize our thinking about sh genes, it is time to reevaluate. Revolutionary Biology explains in simple, vivid terms what this exciting approach has to off er, and then applies its stunning insights to human beings. Th is novel perspective, galvanizes our understanding of how evolution works, what living things are all about and, not least, what it means to be human. Th e controversial disciplines of sociobiology and evolutionary psychology have generated startling insights into longstanding questions concerning the nature and purpose of families, altruism vs. selfi shness, and free will vs. biological determinism. Written by one of its foremost fi gures, Revolutionary Biology is a manifesto and educated layman's guide to this ongoing revolution.

Spacing Constraints on Reinitiation of Paramyxovirus Transcription: The Gene End U Tract Acts as a Spacer to Separate Gene End from Gene Start Sites

The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3′ polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT–PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of ∼250–290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH–HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20–30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.

Recombinant Respiratory Syncytial Viruses with Deletions in the NS1, NS2, SH, and M2-2 Genes Are Attenuated in Vitro and in Vivo

Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: ΔNS1NS2, ΔM2-2SH, ΔM2-2NS2, ΔSHNS1, ΔSHNS2, and ΔSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, ΔM2-2NS2 was most attenuated. ΔM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log10 compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication.

Respiratory syncytial virus infection and G and/or SH protein expression contribute to substance P, which mediates inflammation and enhanced pulmonary disease in BALB/c mice.

A distinct clinical presentation of respiratory syncytial virus (RSV) infection of humans is bronchiolitis, which has clinical features similar to those of asthma. Substance P (SP), a tachykinin neuropeptide, has been associated with neurogenic inflammation and asthma; therefore, we chose to examine SP-induced inflammation with RSV infection. In this study, we examined the production of pulmonary SP associated with RSV infection of BALB/c mice and the effect of anti-SP F(ab)(2) antibodies on the pulmonary inflammatory response. The peak production of pulmonary SP occurred between days 3 and 5 following primary RSV infection and day 1 after secondary infection. Treatment of RSV-infected mice with anti-SP F(ab)(2) antibodies suggested that SP may alter the natural killer cell response to primary and secondary infection. In mice challenged after formalin-inactivated RSV vaccination, SP appears to markedly enhance pulmonary eosinophilia as well as increase polymorphonuclear cell trafficking to the lung. Based on studies with a strain of RSV that lacks the G and SH genes, the SP response to RSV infection appears to be associated with G and/or SH protein expression. These data suggest that SP may be an important contributor to the inflammatory response to RSV infection and that anti-SP F(ab)(2) antibodies might be used to ameliorate RSV-associated disease.

Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees.

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.

Wild type mumps viruses circulating in China establish a new genotype

By analysing the nucleotide sequence of the SH genes of five mumps virus strains derived from the clinical specimens collected during the 1995/96 mumps epidemic in China a new genotype has been established. The circulating viruses showed divergence ranging from 0.8-4.5% at the nucleotide level and 3.5–12.3% at the amino acid level. In addition, a more rational approach has been taken in proposing genotype groupings to MuV strains.

Clustering of mumps virus isolates by SH gene sequence only partially reflects geographical origin.

The nucleotide sequences of the SH gene of 45 new mumps virus isolates derived from different parts of Europe, Canada and USA were determined. A phylogenetic tree was constructed which confirmed the existence of three major clusters. While clustering according to geographical origin was clear in some cases each of the major clusters included isolates which were widely separated in origin. The degree of variability between the sequences of SH genes of different strains and the ratio of coding to non coding differences were both very high compared to those observed in other genes such as the M and HN. A standardized system of nomenclature of mumps strains was established.