Virology Journal - Latest Articles

Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae

Erik Reaves — 2012-05-21T00:00:00Z

Background: Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World Primates. To investigate the suitability of the Aotus nancymaae owl monkey as a viable animal model for rhabdovirus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus.MethodS Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/ V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. Results: Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p=0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies. None of the brain sections exhibited immunoreactivity for rabies virus antibody. DISCUSSION This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. Conclusions: Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek's disease virus.

Ayumi Matsuyama-Kato — 2012-05-21T00:00:00Z

Background: An immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek's disease virus (MDV), which induces immunosuppression in infected chickens. Results: A chicken T cell line, Lee1, which constitutively produces IFN-gamma was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-gamma expression level was analyzed in the cell line by real-time RT-PCR. The IFN-gamma expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field. Conclusions: We demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host.

Computational identification of microRNAs in anatid herpesvirus 1 genome

Jun Xiang — 2012-05-14T00:00:00Z

Background: MicroRNAs (miRNAs) are a group of short (~22 nt) noncoding RNAs that specifically regulate gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of ~70 nt, are processed into mature miRNAs by cellular RNases III. To date, thousands of miRNAs have been identied in different organisms. Several viruses have been reported to encode miRNAs.FindingsHere, we extended the analysis of miRNA-encoding potential to the Anatid herpesvirus 1 (AHV-1). Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs. We then compared the 12 mature miRNAs candidates with the all known miRNAs of the herpesvirus family. Interestingly, the "seed sequences" (nt 2 to 8) of 2 miRNAs were predicted to have the high conservation in position and/or sequence with the 2 miRNAs of Marek's disease virus type 1 (MDV-1). Additionally, we searched the target from viral mRNAs. Surprisingly, none of its own viral transcripts could potentially be targeted. Conclusions: Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs and 2 miRNAs have the high conservation with the 2 miRNAs of MDV-1. The result suggested that AHV-1 and MDV-1 should have closed evolutionary relation, which provides a valuable evidence of classification of AHV-1. Additionally, none of viral gene targets were found, which suggests that AHV-1 miRNAs could not affect its own gene expression.

HHV-8 reduces dendritic cell migration through down-regulation of cell-surface CCR6 and CCR7 and cytoskeleton reorganization

Mara Cirone — 2012-05-14T00:00:00Z

Background: For an efficient immune response against viral infection, dendritic cells (DCs) must express acoordinate repertoire of receptors that allow their recruitment to the sites of inflammation andsubsequently to the secondary lymphoid organs in response to chemokine gradients.Several pathogens are able to subvert the chemokine receptor expression and alter themigration properties of DCs as strategy to escape from the immune control.FindingsHere we report the inhibitory effect of Human Herpesvirus 8 (HHV-8) on the migratorybehavior of immature and mature DCs. We found that the virus altered the DC chemokinereceptor expression and chemokine induced migration. Moreover HHV-8 was also able tointerfere with basal motility of DCs by inducing cytoskeleton modifications. Conclusion: Based on our findings, we suggest that HHV-8 is able to subvert the DC migration capacityand this represents an additional mechanism which interferes with their immune-functions.

Subtype- and antigenic site-specific differences in biophysical influences on evolution of influenza virus hemagglutinin

Stephen Stray — 2012-05-08T00:00:00Z

Background: Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge. Methods: To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures. Results: Areas of HA showing the highest frequency of changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties. Conclusions: The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.

The screening and functional study of proteins binding with the BmNPV polyhedrin promoter

Wei Yu — 2012-05-06T00:00:00Z

Background: The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. Results: In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. Conclusions: Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region.

Characterization of grass carp reovirus minor core protein VP4

Liming Yan — 2012-05-04T00:00:00Z

Background: Grass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of familyReoviridae, contains eleven segmented (double-stranded RNA) dsRNA genome whichencodes 12 proteins. A low-copy core component protein VP4,encoded by the viral genomesegment 5(S5),has been suggested to play a key role in viral genome transcription andreplication. Results: To understand the role of minor core protein VP4 played in molecular pathogenesis duringGCRV infection, the recombinant GCRV VP4 gene was constructed and expressed in bothprokaryotic and mammalian cells in this investigation. The recombinant His-tag fusion VP4products expressed in E.coli were identified by Western blotting utilizing His-tag specificmonoclonal and GCRV polyclonal antibodies. In addition, the expression of VP4 in GCRVinfected cells, appeared in granules structure concentrated mainly in the cytoplasm, can bedetected by Immunofluorescence (IF) using prepared anti-VP4 polyclonal antibody.Meanwhile, VP4 protein in GCRV core and infected cell lysate was identified byImmunoblotting (IB) assay. Of particular note, the VP4 protein was exhibited a diffusedistribution in the cytoplasm and nucleus in transfected cells, suggesting that VP4 proteinmay play a partial role in the nucleus by regulating cell cycle besides its predictedcytoplasmic function in GCRV infection. Conclusions: Our results indicate the VP4 is a core component in GCRV. The cellular localization of VP4is correlated with its predicted function. The data provide a foundation for further studiesaimed at understanding the role of VP4 in viroplasmic inclusion bodies (VIB) formationduring GCRV replication and assembly.

Correction: Hepatitis B virus infection and replication in human bone marrow mesenchymal stem cells

Ruiping Ma — 2012-05-04T00:00:00Z

CorrectionAfter publication of this work (Virol J 2011, 8:486), we noted that D of Figure 1 was incorrect. Now the correct figure has been provided with this correction.

Elevated levels of vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever

Kalichamy Alagarasu — 2012-05-04T00:00:00Z

Background: Altered plasma concentrations of vitamin D and mannose binding lectin (MBL), componentsof innate immunity, have been shown to be associated with the pathogenesis of viralinfections. The objective of the present study was to find out whether plasma concentrationsof MBL and vitamin D are different in patients with dengue fever (DF) and denguehemorrhagic fever (DHF).The resultsThe plasma concentrations of vitamin D and MBL were assessed in 48 DF cases, 45 DHFcases and 20 apparently healthy controls using ELISA based methods. Vitamin Dconcentrations were found to be higher among both DF and DHF cases as compared tohealthy controls (P < 0.005 and P < 0.001). Vitamin D concentrations were not differentbetween DF and DHF cases. When the dengue cases were classified into primary andsecondary infections, secondary DHF cases had significantly higher concentrations ofvitamin D as compared to secondary DF cases (P < 0.050). MBL concentrations were notsignificantly different between healthy controls and dengue cases. MBL concentrations wereobserved to be lower in DHF cases as compared to DF cases (P < 0.050). Although MBLlevels were not different DF and DHF cases based on immune status, the percentage ofprimary DHF cases (50%) having MBL levels lower than 500 ng/ml were less compared toprimary DF cases (P = 0.038). Conclusions: The present study suggests that higher concentrations of vitamin D might be associated withsecondary DHF while deficiency of MBL may be associated with primary DHF.

Protection conferred by a recombinant Marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken

Xiaorong Zhang — 2012-05-04T00:00:00Z

Background: In many countries, the predominant field isolates of infectious bronchitis virus (IBV) have been classified as QX-like strains since 1996. However, no commercial vaccines that are specific for this type of IBV are currently available. Therefore, there is an urgent need to develop novel vaccines that prevent QX-like IBV infection. Results: A recombinant Marek's disease virus (MDV), rMDV-S1, that expresses the S1 subunit of the spike (S) protein from the QX-like infectious bronchitis virus (IBV) was constructed by inserting the IBV S1 gene into the genome of the CVI988/Rispens strain of MDV. Specific pathogen-free (SPF) chickens that were vaccinated with rMDV-S1 were protected when challenged with the QX-like IBV. They were observed to have mild clinical signs of disease, a short virus-shedding period and low mortality. Additionally, the rMDV-S1 conferred full protection to chickens against virulent MDV, as did the CVI988/Rispens strain. Conclusions: Our results demonstrate that rMDV-S1 is an effective and promising recombinant vaccine for the prevention of QX-like IBV infection.