An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

H5N1 avian influenza re-emergence of Lake Communication Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation

Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/ 02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.

Development of a reverse genetics system enabling the rescue of recombinant avian influenza virus A/Turkey/England/50-92/91 (H5N1)

We previously described the use of an established reverse genetics system for the generation of recombinant human influenza A viruses from cloned cDNAs. Here, we have assembled a set of plasmids to allow recovery of the avian H5N1 influenza virus A/Turkey/England/50-92/91 entirely from cDNA. This system enables us to introduce mutations or truncations into the cDNAs to create mutant viruses altered specifically in a chosen gene. These mutant viruses can then be used in future pathogenesis studies in chickens and in studies to understand the host range restrictions of avian influenza viruses in humans.

Presence of antibodies against H5, H7 and H9 influenza a virus in wild birds in the state of São Paulo, Brazil

Although the natural reservoirs of the avian influenza (AI) virus have been extensively studied in many countries, there is a clear lack of information on this subject in South America, particularly in Brazil. The objective of this study was to conduct a serological survey for H5, H7 and H9 antibodies to AI-subtype viruses in wild birds in the state of São Paulo, Brazil. Serum samples were tested using the hemagglutination-inhibition assay. Out of the 31 wild birds sampled between January and December of 2006, seven (22.58%), were seropositive for H5, H7 and H9; four (12.90%) were seropositive for H5 and H7; 13 (41.94%), were seropositive only for H7; three (9.7%), were seropositive only for H9; and four (12.90%) were negative for all three hemagglutinin subtypes. These results indicate that AI viruses belonging to H5, H7 and H9 subtypes circulate among wild birds in the state of São Paulo in the form of either concurrent or consecutive infections. This study contributes to the knowledge of AI epidemiology in Brazil, and stresses the need of further detailed and long-term epidemiological and ecological investigation to determine the current status of this virus.

Aplicação de modelos lineares para análise de expressão gênica em experimentos de microarrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mapping T-cell epitopes of the rubella virus structural proteins

Rubella virus (RV) typically causes a mild childhood illness, but complications can result from both viral and immune-mediated pathogenesis. RV can persist in the presence of neutralizing antibodies, suggesting that cell-mediated immune responses may be necessary for viral clearance. However, the molecular determinants recognized by RV-specific T-cells have not been identified. Using recombinant proteins which express the entire RV structural open reading frame in proliferation assays with lymphocytes of RV-immune individuals, domains which elicit major histocompatibility complex class II-restricted helper T-cells were identified. Synthetic peptides representing these domains were used to define specific epitopes. Two immunodominant domains were mapped to the capsid protein sequence C$\sb1$-C$\sb{29}$ and the E1 glycoprotein sequence E1$\sb{202}$-E1$\sb{283}.$ RV-specific MHC class I-restricted cytotoxic T lymphocytes (CTLs) were identified using a chromium-release assay with infected fibroblasts as target cells. An infectious Sindbis virus vector expressing each of the RV structural proteins identified the capsid, E2 and E1 proteins as targets of CTLs. Specific CTL epitopes were mapped within the previously identified immunodominant domains. This study identified domains of the RV structural proteins that may be beneficial for development of a synthetic vaccine, and provides normative data on RV-specific T-cell responses that should enhance our ability to understand RV persistence and associated complications. ^

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)(1). Assembly of an infectious virion proceeds in two stages(2). In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation(3). However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-angstrom resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus

We describe here the characterization of the gene gp64 encoding the envelope fusion protein GP64 (open reading frame) ORF 105 from Bombyx mori nucleopolyhedrovirus (BmNPV). gp64 was transcribed from the early to late stages of infection and the transcripts were seen from 6 to 72 h post infection (hpi). The early transcripts initiated from a consensus CAGT motif while the late transcripts arose from three conserved TAAG motifs, all of which were located in the near upstream region of the coding sequence. Both early and late transcripts terminated at a run of T residues following the second polyadenylation signal located 31 nt downstream of the translation termination codon. BmGP64 protein was detectable from 6 hpi and was present in larger quantities throughout the infection process from 12 hpi, in BmNPV-infected BmN cells. The persistent presence of GP64 in BmN cells differed from the protein expression pattern of GP64 in Autographa californica multinucleocapsid nucleopolyhedrovirus infection, where the protein levels decreased significantly by late times (48 hpi). BmGP64 was located in the membrane and cytoplasm of the infected host cells and as a component of the budded virions. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited. (C) 2003 Elsevier Science B.V. All rights reserved.

恒河猴TRIM5α的组织分布及不同刺激促进PBMC中TRIM5α转录水平的上调

TRIM5α(tripartite motif protein 5-alpha)蛋白是恒河猴体内一种非常重要的限制因子,能抑制人免疫缺陷病毒(HIV-1,human immunodeficiency virus type 1)、马感染性贫血病毒(EIAV, equine infectious anemia virus)和猫免疫缺陷病毒(FIV, feline immunodeficiencyvirus)等逆转录病毒的复制.恒河猴TRIM5α的组织分布以及在受到外界刺激时TRIM5α mRNA表达量的变化研究还未见报道.本研究从中国恒河猴的各组织中提取总RNA,以β-actin基因作为内参照,通过半定量RT-PCR检测各组织中TRIMSα mRNA的表达.选择HIV-GFP-VSVG假病毒感染外周血单核细胞(peripheral blood mononuclear cell,PBMC),非特异性刺激剂--佛波脂(Phorbol myfismte acetate,PMA)+离子霉素(ionomycin,Ion)及CD28抗体+CD49d抗体分别共刺激恒河猴PBMC,研究不同刺激对恒河猴TRIM5α mRNA表达水平的影响.结果表明:TRIM5α mRNA表达于所研究的恒河猴21种组织中,免疫系统和泌尿生殖系统组织中表达量最高,而神经系统组织,如大脑、脊髓中表达较少,其他组织中未见明显的表达差异;HIV-GFP-VSVG感染和用PMA+Ion、CD28抗体+CD49d抗体分别共刺激PBMC能促进PBMC中TRIM5α mRNA的转录水平的上调.

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4+ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Epidemiological study on antimicrobial use and non-compliance with withdrawal times in broiler farms in Vietnam

L’utilisation d’antimicrobiens chez les animaux de consommation est une source de préoccupation importante pour la santé publique à travers le monde en raison de ses impacts potentiels sur l’émergence de micro-organismes résistants aux antimicrobiens et sur la présence de résidus antimicrobiens néfastes dans la viande. Cependant, dans les pays en développement, peu de données sont disponibles sur les pratiques d’utilisation des antimicrobiens à la ferme. Par conséquent, une étude épidémiologique transversale a été menée de juin à août 2011 dans des élevages de poulets de chair situés dans le sud du Vietnam, ayant pour objectifs de décrire la prévalence d’utilisation des antimicrobiens ajoutés à l’eau de boisson ou aux aliments à la ferme, et de tester les associations entre les caractéristiques des fermes et la non-conformité avec les périodes de retrait recommandés sur l’étiquette des produits. Un échantillon d’accommodement de 70 fermes a été sélectionné. Les propriétaires des fermes ont été interrogés en personne afin de compléter un questionnaire sur les caractéristiques des fermes et les pratiques d’utilisation d’antimicrobiens. Au cours des 6 mois précédant les entrevues, il a été rapporté que la colistine, la tylosine, l’ampicilline, l’enrofloxacine, la doxycycline, l’amoxicilline, la diavéridine et la sulfadimidine ont été utilisés au moins une fois dans les fermes échantillonnées, avec une fréquence descendante (de 75.7% à 30.0%). D’après deux scénarios de risque basés sur la comparaison de la période de retrait recommandée sur l’étiquette du produit et celle pratiquée à la ferme, de 14.3% à 44.3% des propriétaires de ferme interrogés n’ont pas respecté la période de retrait recommandée sur l’étiquette au moins une fois au cours des 6 derniers mois, et ce pour au moins un antimicrobien. Les facteurs de risque associés (p<0.05) avec une non-conformité avec la période de retrait recommandée sur l’étiquette pour au moins un des deux scénarios sont les suivants : élever des oiseaux qui n’appartiennent pas tous à des races d’origine asiatique, vacciner contre la bronchite infectieuse, avoir utilisé plus de 6 différents antimicrobiens à la ferme au cours des 6 derniers mois, et utiliser un mélange d’aliments fait maison et commerciaux. Nos résultats soulignent l’importance d’utiliser les antimicrobiens de façon judicieuse et en respectant les temps de retrait officiels, afin de protéger le consommateur contre les risques pour la santé causés par une exposition à des niveaux nocifs de résidus antimicrobiens.