bluuebunny/crossref_metadata_2025 huggingface dataset:
Re-emergence of chikungunya and o’nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships
Authors: Powers Ann M. , Brault Aaron C. , Tesh Robert B. , Weaver Scott C.
Published: August 2015

Chikungunya (CHIK) virus is a member of the genus Alphavirus in the family Togaviridae. Serologically, it is most closely related to o’nyong-nyong (ONN) virus and is a member of the Semliki Forest antigenic complex. CHIK virus is believed to be enzootic throughout much of Africa and historical evidence indicates that it spread to other parts of the world from this origin. Strains from Africa and Asia are reported to differ biologically, indicating that distinct lineages may exist. To examine the relatedness of CHIK and ONN viruses using genetic data, we conducted phylogenetic studies on isolates obtained throughout Africa and Southeast Asia. Analyses revealed that ONN virus is indeed distinct from CHIK viruses, and these viruses probably diverged thousands of years ago. Two distinct CHIK virus lineages were delineated, one containing all isolates from western Africa and the second comprising all southern and East African strains, as well as isolates from Asia. Phylogenetic trees corroborated historical evidence that CHIK virus originated in Africa and subsequently was introduced into Asia. Within the eastern Africa and southern Africa/Asia lineage, Asian strains grouped together in a genotype distinct from the African groups. These different geographical genotypes exhibit differences in their transmission cycles: in Asia, the virus appears to be maintained in an urban cycle with Aedes aegypti mosquito vectors, while CHIK virus transmission in Africa involves a sylvatic cycle, primarily with Ae. furcifer and Ae. africanus mosquitoes.
Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants
Authors: Krempl C. , Ballesteros M.-L. , Zimmer G. , Enjuanes L. , Klenk H.-D. , Herrler G.
Published: August 2015

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity – accessory binding to intestinal tissues – is also discussed.
Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa
Authors: Cornelissen Marion , van den Burg Remco , Zorgdrager Fokla , Goudsmit Jaap
Published: August 2015

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A
gag
/G
pol
/A
env
, three with A
gag
/G
pol
/G
env
and one other with A
gag
/G
pol
/G
env
, in addition to ‘pure’ subtypes A
gag
/A
pol
/A
env
(n=1) and G
gag
/G
pol
/G
env
(n=4). To analyse the crossover points in more detail, a new RT–PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A
gag
/G
pol
/A
env
recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A
gag
/G
pol
/G
env
recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.
Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens
Authors: Schröder Anja , van Loon Adriaan A. W. M. , Goovaerts Danny , Mundt Egbert
Published: August 2015

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family Birnaviridae. Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3′-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.
Molecular characterization of Kaposi’s sarcoma-associated herpesvirus/human herpesvirus-8 strains from Russia
Authors: Lacoste Vincent , Kadyrova Elena , Chistiakova Irina , Gurtsevitch Vladimir , Judde Jean-Gabriel , Gessain Antoine
Published: August 2015

We report the molecular characterization, with subtyping of both K1 and K14.1/K15 genomic regions, of seven new human herpesvirus-8 (HHV-8) strains from Russian patients with classical Kaposi’s sarcoma. Phylogenetic studies, based on the complete K1 gene/protein analysis, indicate that six of these strains belong to the A subtype, with one belonging to the A4 group and exhibiting a unique deletion of 19 amino acids in the VR2 region at position 186–204. PCR-based studies of the K14.1/K15 genomic region indicate that four of the new strains were of the M subtype while three belonged to the P subtype. Our study indicates an important genetic diversity of the HHV-8 strains currently present in Russia, including a new peculiar strain possessing a unique deletion in the VR2 segment, and confirms the absence of correlation between the K1 and K14.1/K15 molecular subtypes, as M and P genotypes can be observed in the A K1 subtype.
Properties of a neutralizing antibody that recognizes a conformational form of epitope ERDRD in the gp41 C-terminal tail of human immunodeficiency virus type 1
Authors: Cleveland S. Matthew , Jones Tim D. , Dimmock Nigel J.
Published: August 2015

The possibility that epitopes from the C-terminal tail of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) are exposed the surface of the virion has long been contentious. Resolution of this has been hampered by the absence of any neutralizing monoclonal antibodies, but we have recently epitope-purified a neutralizing polyclonal IgG specific for one of the putative gp41 tail epitopes, 746ERDRD750. This was obtained from mice immunized parenterally with a plant virus chimera expressing residues 731–752 from the gp41 tail. The ERDRD epitope is highly conformational and is conserved in 81% of B clade viruses. Here, it is shown that this polyclonal ERDRD-specific IgG is highly potent, with an affinity of 2·2×108 M−1, and a neutralization rate constant (−K
neut) of 7·8×104 M−1 s−1 that exceeds that of nearly all other known HIV-1-neutralizing antibodies. ERDRD-specific IgG gave 50% neutralization at 0·1–0·2 μg/ml and 90% neutralization at approximately 3 μg/ml. It also neutralized virus that was already attached to target cells, and this and other data suggest that it neutralized by inhibiting a virion event that precedes the fusion–entry process. Consistent with this conclusion was the finding that neutralizing amounts of ERDRD-specific IgG did not inhibit the attachment of free virus to target cells. ERDRD-specific IgG was also cross-reactive and neutralized all but one of six B clade T cell line-adapted strains tested.
Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs
Authors: López Nora , Scolaro Luis , Rossi Carlos , Jácamo Rodrigo , Candurra Nélida , Pujol Carlos , Damonte Elsa B. , Franze-Fernández María T.
Published: August 2015

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV–GTac) or the nucleocapsid protein (VV–N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV–GTac or VV–N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV–GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV–GTac survived, whereas all animals inoculated with VV–N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV–GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV–GJun survived lethal JUNV challenge. Protection with either VV–GJun or VV–GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.
Enzymatic properties of hepatitis C virus NS3-associated helicase
Authors: Paolini Chantal , De Francesco Raffaele , Gallinari Paola
Published: August 2015

The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed inEscherichia coliand purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3′ to 5′ direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem–loop RNA structure (SL I) within the 3′-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.
Molecular epidemiology and evolution of coxsackievirus A9
Authors: Santti Juhana , Harvala Heli , Kinnunen Leena , Hyypiä Timo
Published: August 2015

Genetic relationships between 35 clinical isolates of coxsackievirus A9 (CAV9), collected during the last five decades from different geographical regions, were investigated by partial sequencing. Analysis of a 150 nucleotide sequence at the VP1/2A junction region identified 12 CAV9 genotypes. While most of the strains within each genotype showed geographical clustering, the analysis also provided evidence for long-range importation of virus strains. Phylogenetic analysis of a longer region around the VP1/2A junction (approximately 390 nucleotides) revealed that the designated genotypes actually represented phylogenetic lineages. The phylogenetic grouping pattern of the isolates in the analysis of the VP4/VP2 region was similar to that obtained in the VP1/2A region whereas analysis of the 3D region indicated a strikingly different grouping, which suggests that recombination events may occur in the region encoding the nonstructural proteins. Analysis of the deduced amino acid sequences of the VP1 polypeptide demonstrated that the RGD (arginine-glycine-aspartic acid) motif, implicated in the interaction of the virus with integrin, was fully conserved among the isolates.
Inhibition of cap-dependent gene expression induced by protein 2A of hepatitis A virus
Authors: Maltese Elisabetta , Bucci Mauro , Macchia Stefania , Latorre Patrizia , Pagnotti Paolo , Pierangeli Alessandra , Bercoff Raoul Pérez
Published: August 2015

The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760–1087) were linked to the 5′-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in culture supernatants. Expression of HAV peptides extending from aa 764, 776 or 791 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-independent expression of a reporter gene (CAT) directed by the poliovirus RNA 5′-UTR was unaffected. The inhibitory effect was absent in constructs expressing either the short peptide encompassing aa 760–836 or proteins initiated downstream of the putative cleavage site 836–837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791–792 to residue 981, while peptides initiated at the Gln/Ala pair 836–837 may result from alternative cleavage. Point mutations that substituted members of the triad Ser916, His927 and Asp931 abolished the inhibitory effect on cap-dependent translation, suggesting that the HAV-induced CPE may be mediated by 2A protein.
Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene
Authors: Thomas Carole J. , Gooday Graham W. , King Linda A. , Possee Robert D.
Published: August 2015

The chitinase ofAutographa californicanucleopolyhedrovirus (AcMNPV) is required for the characteristic liquefaction of baculovirus-infected insect larvae. Alignments of the putative active sites of a range of chitinases revealed two highly conserved residues, glutamate and aspartate, which have been proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses were generated, AcchiAD311G, AcchiAE315Gand AcchiAD311G E315G, which contained mutations at either the glutamate, the aspartate or both. It was demonstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the attenuation of chitinolytic activity and the cessation of liquefaction ofTrichoplusia nilarvae infected with AcchiAD311G E315G. Mutagenesis of the glutamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, AcchiAE315G, liquefied more slowly than those larvae infected with wild-type AcMNPV. Mutagenesis of the aspartate residue resulted in a reduction of exochitinase activity but an unexpected enhancement of endochitinolytic activity. Liquefaction of AcchiAD311G-infected larvae was observed at the same time as that of AcMNPV-infected larvae.
The rate of progression to AIDS is independent of virus dose in simian immunodeficiency virus-infected macaques
Authors: Holterman Lennart , Niphuis Henk , Koornstra Wim , Dubbes Rob , ten Haaft Peter , Heeney Jonathan L.
Published: August 2015

Of the viral factors that are proposed to influence the rate of progression to AIDS, the role of infectious dose remains unresolved. Intravenous infection of outbredMacaca mulattawith various doses of simian immunodeficiency virus isolate 8980 (SIV8980) revealed an endpoint from which an infectious dose 50 (ID50) was defined. In the six infected animals, the time to develop AIDS was variable with a spectrum of rapid, intermediate and slow progressors. High and sustained plasma viraemia with marked loss of CD4+T-cells was a distinguishing feature between rapid versus intermediate and slow progressors. Animals that received the highest doses did not develop the highest sustained viral loads, nor did they progress more rapidly to disease. Similarly, animals infected with lower doses did not uniformly develop lower viral loads or progress more slowly to AIDS. Furthermore, compiled data from more than 21 animals infected with different doses of the same virus administered by the same route failed to reveal any correlation of infectious dose with survival. Indeed, host factors of these outbred animals, rather than dose of the initial inoculum, were probably an important factor influencing the rate of disease progression in each individual animal. Comparison of animals infected with SIVB670, from which SIV8980was derived, revealed marked differences in disease progression. Clearly, although dose did not influence viral loads nor disease progression, the virulence of the initial inoculum was a major determinant of the rate of progression to AIDS.
The strong positive correlation between effective affinity and infectivity neutralization of highly cross-reactive monoclonal antibody IIB4, which recognizes antigenic site B on influenza A virus haemagglutinin
Authors: Kostolanský F. , Varečková E. , Betáková T. , Mucha V. , Russ G. , Wharton S. A.
Published: August 2015

Monoclonal antibody (MAb) IIB4 displays a rare combination of virus neutralization (VN) activity and broad cross-reactivity with influenza A virus strains of the H3 subtype isolated in a period from 1973 to 1988. The epitope of this antibody has been identified as around HA1 residues 198, 199 and 201. Here we report that residues 155, 159, 188, 189 and 193 also influence the binding of this antibody. We have used this antibody to study the relationship between antibody affinity and VN activity. Using one MAb and a single epitope on the haemagglutinin (HA) of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. A 10-fold increase in effective affinity corresponded to the 2000-fold increase in VN titre. It follows from the law of mass action that for an effective affinityK=9×108 l/mol, 50% VN was achieved at approx. 10% occupation of HA spikes with antibody. In contrast, for an effective affinityK=6×107 l/mol, to achieve 50% VN, occupation of up to 98% of HA spikes was required. An effective affinity aboutK=6×107l/mol thus represents the limiting value for VN because a further decrease in the affinity cannot be compensated by a higher concentration of antibody.
Cross-reactivity of the anti-PML antibody PG-M3 with the herpes simplex virus type 1 immediate early protein ICP4
Authors: Boulware Stefanie L. , Weber Peter C.
Published: August 2015

The PML protein is one of the components of ND10, nuclear matrix-associated structures which undergo rapid disintegration at the onset of herpes simplex virus type 1 (HSV-1) infection. This disruption event has been frequently visualized in immunofluorescence assays using the anti-PML mouse monoclonal antibody PG-M3. This antibody was surprisingly found to also stain nuclear virus replication compartments when employed at higher concentrations. This was shown to be due to an unexpected cross-reactivity of the PG-M3 antibody with the HSV-1 immediate early protein ICP4, a known component of replication compartments. The sequences of ICP4 recognized by PG-M3 were found to map to the extreme amino-terminal end of the protein, which includes a 21 amino acid segment that is partially homologous to the peptide of PML that was used to make PG-M3. These results suggest that PG-M3 may no longer represent an appropriate antibody for use in visualizing the fate of PML and ND10 during HSV-1 infection.
Expression of a Tranosema rostrale polydnavirus gene in the spruce budworm, Choristoneura fumiferana
Authors: Béliveau Catherine , Laforge Marlène , Cusson Michel , Bellemare Guy
Published: August 2015

The endoparasitic wasp Tranosema rostrale (Ichneumonidae) transmits a polydnavirus (PDV) to its host, Choristoneura fumiferana, during oviposition. Unlike most other PDVs examined, the virus of T. rostrale (TrPDV) does not appear to play an important role in suppressing the host cellular immune response. However, it inhibits host metamorphosis. In the present study, TrPDV gene expression was examined in parasitized and virus-injected last-instar caterpillars. Northern analysis with viral DNA as a probe revealed only one detectable mRNA, of about 650 bp. The corresponding cDNA, termed TrV1, was cloned and sequenced and found to encode a protein of 103 amino acids which, following cleavage of the putative signal peptide, has a predicted molecular mass of 9·3 kDa. This protein displays limited similarity to the VHv1.4 cysteine-rich protein from the PDV of Campoletis sonorensis, mostly within the signal peptide region. By using a TrV1-specific probe, the TrV1 gene was localized to segment G of the TrPDV genome. The cuticle and fat body were identified as the principal sites of TrV1 transcription, with little transcription observed in haemocytes and midgut. Western analysis of proteins extracted from selected tissues of parasitized insects suggested that the TrV1 protein is secreted in the haemolymph. As observed for other PDVs, injection of TrPDV did not suppress transcription of the gene that encodes juvenile hormone esterase, the activity of which is inhibited by the virus. We speculate that the TrV1 protein may play a role in the inhibition of C. fumiferana metamorphosis.
Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein
Authors: Hope R. Graham , McLauchlan John
Published: August 2015

From analysis of the primary sequence of the hepatitis C virus (HCV) core protein, we have identified three separable regions based on hydrophobicity and clustering of basic amino acids within the protein. Comparison with capsid proteins of related pesti- and flaviviruses suggested that HCV core has a unique central domain (domain 2). Previous findings have revealed that core protein can associate with lipid droplets which are intracellular storage sites for triacylglycerols and cholesterol esters. Confocal analysis of variant forms lacking regions of core indicated that most residues within the unique region are necessary for association of the protein with lipid droplets. A segment within domain 2 (from residues 125 to 144) also was required for stability of the protein and a polypeptide lacking these sequences was degraded apparently by the proteasome. In cells depleted of lipid droplets, core protein remained located in the cytoplasm. Moreover, cleavage of the protein at the maturation site and stability were not affected by inability to bind to lipid droplets.
Phylogenetic analysis of influenza C virus nonstructural (NS) protein genes and identification of the NS2 protein
Authors: Alamgir A. S. M. , Matsuzaki Yoko , Hongo Seiji , Tsuchiya Emi , Sugawara Kanetsu , Muraki Yasushi , Nakamura Kiyoto
Published: August 2015

The nucleotide sequences of RNA segment 7 (nonstructural protein gene; NS) were compared among 34 influenza C virus strains isolated between 1947 and 1992. The results showed that all the NS genes analysed had the potential to encode NS1 and NS2 proteins of 246 and 182 amino acids, respectively. The deduced amino acid sequence of the previously unidentified NS2 was fairly well conserved, although it was more divergent than the NS1 protein sequence. Moreover, immunoprecipitation experiments with rabbit immune serum against a glutathione S-transferase fusion protein containing the C-terminal region of the 182 amino acid NS2 protein revealed synthesis of a protein with an apparent molecular mass of ∼22 kDa in infected cells. A phylogenetic analysis showed that the 34 NS genes were split into two distinct groups, A and B. Comparison of the phylogenetic positions of the individual isolates in the NS gene tree with those in the haemagglutinin–esterase (HE) gene tree suggested that most of the influenza C viruses currently circulating in Japan, irrespective of their HE gene lineage, had acquired group B NS genes through reassortment events that presumably occurred either in the 1970s or in the early 1980s.
NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2
Authors: Horscroft Nigel J. , Roy Polly
Published: August 2015

The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca2+, Mg2+and Mn2+were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.
Sequence analysis of the long control region of human papillomavirus type 16 variants and functional consequences for P97 promoter activity
Authors: Kämmer Christina , Warthorst Ursula , Torrez-Martinez Norah , Wheeler Cosette M. , Pfister Herbert
Published: August 2015

Genital human papillomaviruses (HPV) are considered to be one of the main risk factors for the development of cervical cancer. The P97 promoter at the E6-proximal end of the long control region (LCR) regulates the transcription of viral genes, especially the oncogenes E6 and E7. The LCR contains binding sites of several viral and cellular transcription factors, which either activate or repress the P97 promoter. Intratype variants of HPV-16 belong to six geographically clustered phylogenetic groups distributed all over the world. These variants exhibit differences in E6 protein activities and in tumour progression in vivo. Seven HPV-16 variants were investigated by sequencing the entire LCR (nt 7060–124) and by comparing the transcriptional activities of their P97 promoters. Previously unknown nucleotide variations were identified in all LCRs investigated. In luciferase assays, 3·3- and 2·8-fold increases in P97 promoter activity were detected in the Asian American c and North American 1 variants when compared with the European reference clone. The African variants 1a and 2a exhibited P97 promoter activities comparable to the European reference clone. After recombining different LCR fragments, the region responsible for enhanced transcription in the Asian American c and North American 1 variants could be attributed to the E6-proximal end of the LCR (nt 7619–124).
Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours
Authors: Sastre-Garau Xavier , Favre Michel , Couturier Jérôme , Orth Gérard
Published: August 2015

We previously described two genital carcinomas (IC2, IC4) containing human papillomavirus type 16 (HPV-16)- or HPV-18-related sequences integrated in chromosomal bands containing the c-myc (8q24) or N-myc (2p24) gene, respectively. The c-myc gene was rearranged and amplified in IC2 cells without evidence of overexpression. The N-myc gene was amplified and highly transcribed in IC4 cells. Here, the sequence of an 8039 bp IC4 DNA fragment containing the integrated viral sequences and the cellular junctions is reported. A 3948 bp segment of the genome of HPV-45 encompassing the upstream regulatory region and the E6 and E7 ORFs was integrated into the untranslated part of N-myc exon 3, upstream of the N-myc polyadenylation signal. Both N-myc and HPV-45 sequences were amplified 10- to 20-fold. The 3′ ends of the major N-myc transcript were mapped upstream of the 5′ junction. A minor N-myc/HPV-45 fusion transcript was also identified, as well as two abundant transcripts from the HPV-45 E6–E7 region. Large amounts of N-myc protein were detected in IC4 cells. A major alteration of c-myc sequences in IC2 cells involved the insertion of a non-coding sequence into the second intron and their co-amplification with the third exon, without any evidence for the integration of HPV-16 sequences within or close to the gene. Different patterns of myc gene alterations may thus be associated with integration of HPV DNA in genital tumours, including the activation of the protooncogene via a mechanism of insertional mutagenesis and/or gene amplification.
Contribution of bovine papillomavirus type 1 E1 protein residue 48 to replication function
Authors: McShan Gina D. , Wilson Van G.
Published: August 2015

The E1 protein of bovine papillomavirus type 1 (BPV-1) is the origin recognition protein and is essential for the initiation of viral DNA replication. We reported previously that there is a conserved motif between residues 25 and 60 of all papillomavirus E1 proteins that resembles a casein kinase II (CKII) phosphorylation site. The corresponding serine in BPV-1, serine-48, is an efficient substrate for CKII in vitro. To examine the functional role of this potential phosphorylation site, three amino acid substitutions were constructed at serine-48. Conversion of serine-48 to a glycine (S48G) resulted in a BPV-1 genome that was unable to replicate and had reduced transformation capacity. The S48G E1 protein also failed to support replication of a BPV-1 origin-containing plasmid when expressed from a heterologous vector rather than the viral genome, indicating a direct replication defect. In contrast, conversion of serine-48 to acidic residues (S48D or S48E), which mimic the charge and structure of phosphoserine, maintained the wild-type replication phenotype. These mutational results are consistent with a replication requirement for a negative charge at serine-48, presumably supplied by in vivo phosphorylation. The mechanistic basis for the negative charge requirement was examined by testing several activities of the S48G mutant E1 protein in vivo using yeast one- and two-hybrid systems. No gross defect was observed for stability, origin binding or interaction with E2 or for E1–E1 interaction, although subtle defects in these activities would not likely be detected. Overall, the results suggest that important phosphoregulatory control of E1 replication function is mediated through the N-terminal region of this protein.
Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex
Authors: Westra Douwe F. , Verjans Georges M. G. M. , Osterhaus Albert D. M. E. , van Kooij Adriaan , Welling Gjalt W. , Scheffer Albert Jan , The T. Hauw , Welling-Wester Sytske
Published: August 2015

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)Ht(His):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy adults. In addition, responses to HSV-1 gDtwere determined. As antigens, purified soluble recombinant forms of the gHt(His):gL complex produced by insect cells and of gDtproduced by yeast cells were used. In contrast to seronegative donors, sera of all seropositive donors contained gHt(His): gL-specific IgG. Using peripheral blood (PB) T cells, gHt(His):gL-specific proliferative T cell responses were detected in all seropositive donors. Culture supernatants of PB T cells stimulated with recombinant gHt(His):gL contained high levels of interferon-γ and no detectable interleukin-4, indicating their Th1 phenotype. These results show that naturally acquired HSV-1 infection induces gH:gL-specific humoral and T cell responses.
On the control of late gene expression in Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8)
Authors: Chang Jean , Ganem Don
Published: August 2015

Herpesvirus late genes require viral DNA replication for maximal expression. Although late gene expression appears to require DNA replication in cis in alphaherpesviruses, studies in Epstein–Barr virus (EBV) suggest that this cis-requirement might not pertain to the gammaherpesviruses. Based on these findings, a system was created to investigate the elements required for the regulation of Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus-8) late gene expression. The transcript of a classic late gene encoding the viral assembly protein was characterized and reporter genes driven by the assembly protein promoter region were constructed. Unlike the EBV case, expression of a reporter gene under the control of the assembly protein promoter did not display authentic regulation when removed from the context of the viral genome. Although reporter expression rose in cells displaying lytic replication, this expression was not diminished by specific inhibitors of viral DNA synthesis. Minimal core promoters were similarly unable to reproduce late gene regulation. These results suggest that proper KSHV late gene expression is likely to be dependent upon virus lytic replication in cis and indicate that the regulation of KSHV late genes more closely resembles that observed in herpes simplex virus than that described for EBV.
Distribution of chicken anaemia virus in the reproductive tissues of specific-pathogen-free chickens
Authors: Cardona Carol J. , Oswald Wendelien B. , Schat K. A.
Published: August 2015

The specific-pathogen-free (SPF) flocks of chickens maintained by the Department of Microbiology and Immunology at Cornell University became infected, inadvertently, with chicken anaemia virus (CAV), as demonstrated by seroconversion. Chickens from five flocks representing three different strains were examined for the presence of CAV using nested PCR. Virus was detected in ovaries, infundibula, vas deferentia, testes and spleens. Ovaries were positive in 38 to 72% of the hens in four flocks with 13 to 56 birds examined per flock. Interestingly, the ovaries were often the only positive tissues, while a few hens had only positive spleens. In roosters, the vas deferens was positive in 30 to 79% of the birds with 5 to 19 birds examined per flock; the vas deferens was the only positive tissue in 20 to 37%. Individual cells in the theca externa and rare epithelial cells in the infundibular epithelium were positive for CAV by in situ PCR. Positive cells were not detected in testes or vas deferentia. The SH-1 strain of CAV was isolated from these tissues and partially sequenced. Only minor sequence differences were found compared to CIA-1 and Cux-1. Embryos from matings between persistently infected dams and sire had CAV-positive cells in mesenchyme near the developing vertebral column. The data show that CAV persists in the reproductive tissues far longer than previously thought, and that it can be vertically transmitted from persistently infected birds.
Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein
Authors: Satoh Hiroshi , Matsuda Hironori , Kawamura Takehiro , Isogai Masamichi , Yoshikawa Nobuyuki , Takahashi Tsuyoshi
Published: August 2015

The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP–GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP–GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP–GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287–457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.
Vaccination of cattle with attenuated rinderpest virus stimulates CD4+ T cell responses with broad viral antigen specificity
Authors: Lund Brett T. , Tiwari Ashok , Galbraith Sareen , Baron Michael D. , Morrison W. Ivan , Barrett Tom
Published: August 2015

The immune responses of cattle inoculated with either a virulent or an attenuated vaccine strain of rinderpest virus (RPV) were examined by measuring the proliferation of peripheral blood mononuclear cells (PBMC) to whole RPV antigen preparations and to individual RPV major structural proteins expressed using recombinant adenoviruses. Responses to the T cell mitogen concanavalin A (ConA) were also measured as a control to monitor non-specific effects of infection with RPV on T cell responses. Infection with the vaccine strain of RPV was found to induce a strong CD4+T cell response. A specific response was detected to all RPV proteins tested, namely the haemagglutinin (H), fusion (F), nucleocapsid (N) and matrix (M) proteins, in animals vaccinated with the attenuated strain of the virus. No one protein was found to be dominant with respect to the induction of T cell proliferative responses. As expected, vaccination of cattle with an unrelated virus vaccine, a capripox vaccine, failed to produce a response to RPV antigens. While profound suppression of T cell responses was observed following infection with the virulent strain of RPV, no evidence of impairment of T cell responsiveness was observed following RPV vaccination, or on subsequent challenge of vaccinated animals with virulent virus.
Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences
Authors: Billecocq A. , Vazeille-Falcoz M. , Rodhain F. , Bouloy M.
Published: August 2015

Rift Valley fever virus (RVFV) is an arbovirus of the Bunyaviridae family, causing recurrent disease outbreaks in Africa. Natural vertebrate hosts include cattle and humans. Several mosquito species belonging to the Aedes and Culex generaact as vectors of this phlebovirus. To test whether pathogen-derived resistance against RVFV could be induced by expressing genomic sequences in mosquito cells, as has been shown for La Crosse and dengue 2 viruses, we generated various recombinant Semliki Forest viruses expressing the S segment (or its genes) in the genomic or antigenomic sense. Expression of the N but not the NSs gene interfered with the production of RVFV in mosquito cells and this phenomenon was RNA- but not protein-dependent. These results raise questions on the molecular mechanisms involved in virus resistance.
Manifestation of thrombocytopenia in dengue-2-virus-infected mice
Authors: Huang Kao-Jean , Li Shu-Yi J. , Chen Shiour-Ching , Liu Hsiao-Sheng , Lin Yee-Shin , Yeh Trai-Ming , Liu Ching-Chuan , Lei Huan-Yao
Published: August 2015

Dengue virus infection causes dengue fever, dengue haemorrhagic fever and dengue shock syndrome. No animal model is available that mimics these clinical manifestations. In this study, the establishment is reported of a murine model for dengue virus infection that resembles the thrombocytopenia manifestation. Dengue-2 virus (dengue virus type 2) can infect murine cells either in vitro (primary cell culture) or in vivo. Viraemia detected by RT–PCR was found transiently at 2 days after intravenous injection of dengue-2 virus. Transient thrombocytopenia developed at 10–13 days after primary or secondary infection. Anti-platelet antibody was generated after dengue-2 virus infection. There was strain variation in dengue-2 virus infection; the A/J strain was more sensitive than BALB/c or B6 mice. This dengue-2-virus-infected mouse system accompanied by thrombocytopenia and anti-platelet antibody will be a valuable model to study the pathogenicity of dengue virus infection.
Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor
Authors: Sbardellati Andrea , Scarselli Elisa , Amati Viviana , Falcinelli Sabrina , Kekulé Alexander S. , Traboni Cinzia
Published: August 2015

The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication. In this report, GBV-B NS3 activity on GBV-B precursor proteins has been analysed in a cell-based system. It is shown that mature protein products are obtained that are compatible with the cleavage sites proposed on the basis of sequence homology with HCV and that GBV-B NS4A protein is required as a cofactor for optimal enzymatic activity. Experiments in vitro supported by a structural model mapped the region of NS4A that interacts with NS3 and showed that the GBV-B cofactor cannot be substituted for by its HCV analogue.
Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins
Authors: Londrigan Sarah L. , Hewish Marilyn J. , Thomson Melanie J. , Sanders Georgina M. , Mustafa Huseyin , Coulson Barbara S.
Published: August 2015

Rotavirus replication occursin vivoin intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins α2β1, α4β1 and αXβ2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed α2β1 and (when tested) αXβ2, whereas the non-permissive K562 cells did not express α2β1, α4β1 or αXβ2. Only RD cells expressed α4β1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface α2 integrin correlated with levels of rotavirus growth. The α2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0·1, and the data best fitted a sigmoidal dose–response curve (r2=1·00,P=0·005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of α2β1 integrin and is consistent with their expression of αXβ2 and α4β1 integrins.
The carboxy terminus of the herpesvirus saimiri ORF 57 gene contains domains that are required for transactivation and transrepression
Authors: Goodwin Delyth J. , Hall Kersten T. , Giles Mathew S. , Calderwood Michael A. , Markham Alex F. , Whitehouse Adrian
Published: August 2015

Herpesvirus saimiri (HVS) ORF 57 is homologous to genes identified in all classes of herpesviruses. We have previously shown that ORF 57 encodes a multifunctional protein, responsible for both transactivation and repression of viral gene expression at a post-transcriptional level. This suggests that the ORF 57 protein shares some functional similarities with the herpes simplex virus IE63/ICP27 and Epstein–Barr virus Mta proteins. However, little is known about the functional domains responsible for the properties of ORF 57 due to the limited homology shared between these proteins. In this report, we have identified the functional domains responsible for transactivation and repression by the ORF 57 protein. We demonstrate that the carboxy terminus is required for ORF 57 transactivation, repression and an intense SC-35 nuclear spotting. This region contains two highly conserved motifs amongst its homologues, a zinc finger-like motif and a highly hydrophobic domain. We further show that the hydrophobic domain is required for transactivation and is also involved in nuclear localization of the ORF 57 protein, whereas the zinc finger-like domain is required for transactivation, repression and the intense SC-35 nuclear spotting.
Characterization of a highly divergent TT virus genome
Authors: Hallett R. L. , Clewley J. P. , Bobet F. , McKiernan P. J. , Teo C. G.
Published: August 2015

A novel TT virus (TTV)-like DNA sequence was detected in the serum of a patient (PM) with acute non-A–E hepatitis. The full-length genome sequence, referred to here as PM virus (PMV), was obtained and its relationship to other full or near full-length TTV sequences examined. Although it shares a common genomic arrangement and short conserved regions, the majority of the genome is extremely divergent, displaying an average genetic distance of 0·60 from all other TTV sequences. By comparing PMV with TTV genomes representing the most divergent types so far described, six major groups can be distinguished. The level of genetic diversity seen between these genomes is higher than would be expected within a single virus species. Indeed, PMV could be considered the prototype of an independent taxonomic group within the Circoviridae family. A genoprevalence study of sera from blood donors and patients with acute hepatitis suggests that PMV is rare.