The development of poultry farms risk assessment tool for avian influenza in Imo State, Nigeria

Background: Nigeria was one of the 13 countries where avian influenza outbreak in poultry farms was reported during the 2006 avian influenza pandemic threat and was also the first country in Africa to report the presence of H5N1influenza among its poultry population. There are multiple hypotheses on how the avian influenza outbreak of 2006 was introduced to Nigeria, but the consensus is that once introduced, poultry farms and their workers were responsible for 70% of the spread of avian influenza virus to other poultry farms and the population. ^ The spread of avian influenza has been attributed to lack of compliance by poultry farms and their workers with poultry farm biosecurity measures. When poultry farms fail to adhere to biosecurity measures and there is an outbreak of infectious diseases like in 2006, epidemiological investigations usually assess poultry farm biosecurity—often with the aid of a questionnaire. Despite the importance of questionnaires in determining farm compliance with biosecurity measures, there have been few efforts to determine the validity of questionnaires designed to assess poultry farms risk factors. Hence, this study developed and validated a tool (questionnaire) that can be used for poultry farm risk stratification in Imo State, Nigeria. ^ Methods: Risk domains were generated using literature and recommendations from agricultural organizations and the Nigeria government for poultry farms. The risk domains were then used to develop a questionnaire. Both the risk domain and questionnaire were verified and modified by a group of five experts with a research interest in Nigeria's poultry industry and/or avian influenza prevention. Once a consensus was reached by the experts, the questionnaire was distributed to 30 selected poultry farms in Imo State, Nigeria that participated in this study. Survey responses were received for all the 30 poultry farms that were selected. The same poultry farms were visited one week after they completed the questionnaires for on-site observation. Agreement among survey and observation results were analyzed using a kappa test and rated as poor, fair, moderate, substantial, or nearly perfect; and internal consistency of the survey was also computed. ^ Result: Out of the 43 items on the questionnaire, 32 items were validated by this study. The agreement between the survey result and onsite observation was analyzed using kappa test and ranged from poor to nearly perfect. Most poultry farms had their best agreements in the contact section of the survey. The least agreement was noted in the farm management section of the survey. Thirty-two questions on the survey had a coefficient alpha > 0.70, which is a robust internal consistency for the survey. ^ Conclusion: This study developed 14 risk domains for poultry farms in Nigeria and validated 32 items from the original questionnaire that contained 43 items. The validated items can be used to determine the risk of introduction and spread of avian influenza virus in poultry farms in Imo State, Nigeria. After further validations in other states, regions and poultry farm sectors in Nigeria; this risk assessment tool can then be used to determine the risk profile of poultry farms across Nigeria.^

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml.

Identification of a cellular receptor for subgroup E avian leukosis virus

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the ΔHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with ΔHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The ΔHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Aminoacylation identity switch of turnip yellow mosaic virus RNA from valine to methionine results in an infectious virus.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.

Expression of transduced genes in mice generated by infecting blastocysts with avian leukosis virus-based retroviral vectors.

Transgenic mouse lines have been developed that express the tv-a receptor under the control of the chicken beta-actin promoter. These mice express the tv-a receptor in most or all tissues and in the early embryo. An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos from these transgenic lines. Experimental animals could be generated quickly and easily by infecting susceptible blastocysts with ALV-based retroviral vectors. Expression of the delivered genes was controlled by either the constitutive viral promoter contained in the long terminal repeat or an internal nonviral tissue-specific promoter. Mating the infected founder chimeric animals produced animals that carry the ALV provirus as a transgene. A subset of the integrated proviruses expressed the chloramphenicol acetyltransferase reporter gene from either the promoter in the long terminal repeat or an internal promoter, which we believe indicates that many of the sites that are accessible to viral DNA insertion in preimplantation embryos are incompatible with expression in older animals. This approach should prove useful for studies on murine cell lineage and development, providing models for studying oncogenesis, and testing gene therapy strategies.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.