- Volume 97, Issue 7, 2016

Puumala hantavirus (PUUV) is the most common and widespread hantavirus in Europe and is associated with a mild form of haemorrhagic fever with renal syndrome in humans, called nephropathia epidemica. This study presents the molecular characterization of PUUV circulating in bank voles in two regions of the Netherlands. Most human cases of hantavirus infection are from these two regions. Phylogenetic analysis of the (partial) S, M and L-segments indicated that the Dutch strains belong to the CE lineage, which includes PUUV strains from France, Germany and Belgium. We have identified two distinct groups of PUUV, corresponding with their geographic origin and with adjoining regions in neighbouring countries.

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25–30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Dengue virus infects immune cells, including monocytes, macrophages and dendritic cells (DC). We compared virus infectivity in macrophages and DC, and found that the virus origin determined the cell tropism of progeny virus. The highest efficiency of re-infection was seen for macrophage-derived dengue virus. Furthermore, in the presence of enhancing antibodies, macrophage-derived virus gave greater enhancement of infection compared with immature DC-derived virus. Taken together, our results highlight the importance of macrophages in dengue infection.

GB virus C (GBV-C) is a non-pathogenic flavivirus that may play a role in modulating HIV disease. Multiple genotypes of GBV-C that have been identified to date that may differentially regulate HIV; however, the number of complete GBV-C sequences published to date is very limited. We sequenced full-length GBV-C genomes from four individuals with HIV/HCV co-infection in the United States. Intergenotypic recombination was evident in two of these individuals. Evaluation of additional full-length GBV-C genomes would facilitate the creation of full-length, replication-competent molecular clones of GBV-C to evaluate the phenotypic diversity of GBV-C genotypes and provide important molecular data on this understudied virus.

A natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus was isolated from an acute flaccid paralytic case in Brazil. Genome sequencing revealed the uncommon location of the crossover site in the VP1 coding region (nucleotides 3251–3258 of Sabin 3 genome). The Sabin 2 donor sequence replaced the last 118 nt of VP1, resulting in the substitution of the complete antigenic site IIIa by PV2-specific amino acids. The low overall number of nucleotide substitutions in P1 region indicated that the predicted replication time of the isolate was about 8–9 weeks. Two of the principal determinants of attenuation in Sabin 3 genomes were mutated (U472C and C2493U), but the temperature-sensitive phenotype of the isolate was preserved. Our results support the theory that there exists a PV3/PV2 recombination hotspot site in the tail region of the VP1 capsid protein and that the recombination may occur soon after oral poliovirus vaccine administration.

Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.

Foot-and-mouth disease (FMD) has a major economic impact throughout the world and is a considerable threat to food security. Current FMD virus (FMDV) vaccines are made from chemically inactivated virus and need to contain intact viral capsids to maximize efficacy. FMDV exists as seven serotypes, each made up by a number of constantly evolving subtypes. A lack of immunological cross-reactivity between serotypes and between some strains within a serotype greatly complicates efforts to control FMD by vaccination. Thus, vaccines for one serotype do not afford protection against the others, and multiple-serotype-specific vaccines are required for effective control. The FMDV serotypes exhibit variation in their thermostability, and the capsids of inactivated preparations of the O, C and SAT serotypes are particularly susceptible to dissociation at elevated temperature. Methods to quantify capsid stability are currently limited, lack sensitivity and cannot accurately reflect differences in thermostability. Thus, new, more sensitive approaches to quantify capsid stability would be of great value for the production of more stable vaccines and to assess the effect of production conditions on vaccine preparations. Here we have investigated the application of a novel methodology (termed PaSTRy) that utilizes an RNA-binding fluorescent dye and a quantitative (q)PCR machine to monitor viral genome release and hence dissociation of the FMDV capsid during a slow incremental increase in temperature. PaSTRy was used to characterize capsid stability of all FMDV serotypes. Furthermore, we have used this approach to identify stabilizing factors for the most labile FMDV serotypes.

Avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (IBV) that affects poultry production worldwide. The absence of vaccine cross-protection and the frequent emergence of new variant strains complicate control of IBV. Here we designed a study to measure the evolution dynamics of IBV strains in China. One hundered and seven complete sequences and 1022 S1-region sequences of Chinese IBVs isolated between 1994 and 2014 were analysed by using MEGA 5.0 software and the Bayesian analysis sampling trees (BEAST) method, and selection pressure on different proteins was assessed. The phylogenetic dissimilarity of different gene trees in the data set indicated possible recombination. Fourteen isolates were identified as recombinants, possibly generated from vaccines of the Massachusetts serotype in recombination with circulating viruses. The earliest IBV in China was found to have existed in the early 1900s, and continues to evolve at a rate of approximately 10− 5 substitutions per site per year. We found that purifying selection was the main evolutionary pressure in the protein-coding regions, while the S1 gene bears the greatest positive selection pressure. The proportion of QX-like genotype strains increased over time. These results indicate that the genotypes of Chinese IBVs have undergone a remarkable transition during the past 20 years.

Passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells resulted in the segmentation of the viral genome into two defective RNAs lacking part of either the L- or the capsid-coding region. The two RNAs are infectious by complementation. Electroporation of L-defective RNA in BHK-21 cells resulted in the accumulation of the precursor P3 located away from the deleted sequence. Expression of L in trans led to the processing of P3, indicating that there is a connection between L protease activity and the secondary cleavages carried out by 3C protease within P3. These results suggest that the complementation mechanism between defective RNAs is not restricted to supplying the L and capsid proteins but that distance effects on polyprotein processing events are also implicated.

Dengue virus (DENV) replication is known to prevent maturation of infected dendritic cells (DCs) thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) influence the phenotype of DCs remains elusive. Here, we evaluated the maturation state and cytokine profile of immDCs exposed to DENV-ICs. Indeed, DENV infection of immDCs in the absence of antibodies was hallmarked by blunted upregulation of CD83, CD86 and the major histocompatibility complex molecule HLA-DR. In contrast, DENV infection in the presence of neutralizing antibodies triggered full DC maturation and induced a balanced inflammatory cytokine response. Moreover, DENV infection under non-neutralizing conditions prompted upregulation of CD83 and CD86 but not HLA-DR, and triggered production of pro-inflammatory cytokines. The effect of DENV-IC was found to be dependent on the engagement of FcγRIIa. Altogether, our data show that the presence of DENV-IC alters the phenotype and cytokine profile of DCs.

Bufavirus is a single-stranded DNA virus belonging to the genus Protoparvovirus. This study reports the identification and characterization of a porcine bufavirus by a metagenomic approach, and a limited epidemiology investigation of bufavirus in six swine farms. A comparative genome analysis showed a similarity of 93 % to a Hungarian porcine bufavirus. Bayesian and maximum-likelihood analyses of genome sequences showed a close relationship of porcine bufaviruses to human and monkey bufaviruses. Molecular dating of the most recent common ancestors supported a recent introduction of bufaviruses into human and pig populations, respectively. A real-time PCR method was developed to screen 60 faecal samples for the porcine bufavirus DNA, and eight positive samples were found in two neighbouring farms, suggesting a relatively low prevalence (13.3 %). No direct transmission of porcine bufaviruses between two neighbouring farms was found, suggesting that bufaviruses may have spread widely in different geographical regions.

The non-coding control region (NCCR) of polyomaviruses includes the promoters for early and late genes, a transcription enhancer and the origin of DNA replication. Particularly virulent variants of the human pathogens BKPyV and JCPyV, as well as of simian virus 40 (SV40), occur in vitro and in vivo. These strains often harbour rearrangements in their NCCR, typically deletions of some DNA segment(s) and/or duplications of others. Using an SV40-based model system we provide evidence that duplications of enhancer elements, whether from SV40 itself or from the related BKPyV and JCPyV, increase early gene transcription and replicative capacity. SV40 harbouring subsegments of the strong cytomegalovirus (HCMV) enhancer replicated better than the common ‘wild-type’ SV40 in the human cell lines HEK293 and U2OS. In conclusion, replacing the SV40 enhancer with heterologous enhancers can profoundly influence SV40’s infective capacity, underscoring the potential of small DNA viruses to overcome cell type and species barriers.

Within the family Adenoviridae, presently Simian mastadenovirus A is the single species approved officially for monkey adenoviruses (AdVs), whilst the establishment of six further species (Simian mastadenovirus B to Simian mastadenovirus G) has been proposed in the last few years. We examined the genetic content and phylogenetic relationships of four Old World monkey (OWM) AdV types [namely simian AdV (SAdV)-8, -11, -16 and -19] for which it had been proposed that they should be classified into different AdV species: SAdV-11 to Human mastadenovirus G, and the other three viruses into three novel species. By full genome sequencing, we identified gene contents characteristic for the genus Mastadenovirus. Among the 36 ORFs, 2 genes of different lengths, predicted to encode the adenoviral cellular attachment protein (the fibre), were found. The E3 regions contained six genes, present in every OWM AdV, but lacked the E3 19K gene, which has seemingly appeared only in the ape (hominid) AdV lineages during evolution. For the first time in SAdVs, two other exons belonging to the gene of the so-called U exon protein were also predicted. Phylogenetic calculations, based on the fibre-1 and the major capsid protein, the hexon, implied that recombination events might have happened between different AdV species. Phylogeny inference, based on the viral DNA-dependent DNA polymerase and the penton base protein, further supported the species classification proposed earlier.

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the ‘a’ determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9–11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs.

Porcine circovirus type 2 (PCV2) is the primary aetiological agent of porcine circovirus-associated disease in swine. The mechanism of PCV2 pathogenesis remains largely unknown. A newly identified viral protein of PCV2, ORF4, has been suggested to be involved in virus-induced apoptosis. However, there is still no information regarding the molecular mechanism by which ORF4 regulates apoptosis. In this study, we reveal that a physical interaction between the PCV2 ORF4 protein and ferritin heavy chain (FHC) in the cytoplasm of host cells reduced the cellular concentration of FHC. The ORF4-mediated reduction of FHC inhibited reactive oxygen species accumulation in PCV2-infected cells. Consequently, the ORF4 protein inhibited apoptosis in host cells. This may be the first report to describe the mechanism of ORF4 cytoprotection against apoptosis during the early stages of PCV2 infection.

Seroepidemiological studies showed that the human polyomavirus KI (KIPyV) is common in the human population, with age-specific seroprevalence ranging from 40–90 %. Genome epidemiological analyses demonstrated that KIPyV DNA is predominantly found in respiratory tract samples of immunocompromised individuals and children suffering from respiratory diseases, but viral sequences have also been detected in brain, tonsil, lymphoid tissue studies, plasma, blood and faeces. Little is known about the sequence variation in the non-coding control region of KIPyV variants residing in different sites of the human body and whether specific strains dominate in certain parts of the world. In this study, we sequenced the non-coding control region (NCCR) of naturally occurring KIPyV variants in nasopharyngeal samples from patients with respiratory symptoms or infection and in blood from healthy donors in Norway. In total 86 sequences were obtained, 44 of which were identical to the original isolated Stockholm 60 variant. The remaining NCCRs contained one or several mutations, none of them previously reported. The same mutations were detected in NCCRs amplified from blood and nasopharyngeal samples. Some patients had different variants in their specimens. Transient transfection studies in HEK293 cells with a luciferase reporter plasmid demonstrated that some single mutations had a significant effect on the relative early and late promoter strength compared with the Stockholm 60 promoter. The effect of the NCCR mutations on viral replication and possible virulence properties remains to be established.

Mastomys coucha, an African rodent, is a useful animal model of papillomavirus infection, as it develops both premalignant and malignant skin tumors as a consequence of a persistent infection with Mastomys natalensis papillomavirus (MnPV). In this study, we mapped the MnPV transcriptome in productive lesions by both classical molecular techniques and high-throughput RNA sequencing. Combination of these methods revealed a complex and comprehensive transcription map, with novel splicing events not described in other papillomaviruses. Furthermore, these splicing occurrences could potentially lead to the expression of novel E2, E1∧E4, E7 and L2 isoforms. Expression level estimation of each transcript showed that late-region mRNAs considerably outnumber early transcripts, with species coding for L1 and E1∧E4 being the most abundant. In summary, the full transcription map assembled in this study will allow us to further understand MnPV gene expression and the mechanisms that lead to natural tumour development.