- Volume 96, Issue 7, 2015

Classical swine fever is one of the most important swine diseases worldwide and has tremendous socioeconomic impact. In this study, we focused on the signalling pathways of Toll-like receptors (TLRs) because of their roles in the detection and response to viral infections. To this end, two classical swine fever virus (CSFV) strains, namely the highly virulent CSFV Shimen strain and the avirulent C strain (a vaccine strain), were employed, and the expression of 19 immune effector genes was analysed by real-time PCR, Western blot analyses, ELISA and flow cytometry analyses. In vitro experiments were conducted with porcine monocyte-derived macrophages (pMDMs). The results showed that the mRNA and protein levels of TLR2, TLR4 and TLR7 were upregulated in response to CSFV infection, but TLR3 remained unchanged, and was downregulated after infection with the C strain and the Shimen virus, respectively. Furthermore, TLR3-mediated innate immune responses were inhibited in Shimen-strain-infected pMDMs by stimulation with poly(I : C). Accordingly, comprehensive analyses were performed to detect TLR-dependent cytokine responses and the activation of TLR signalling elements. CSFV infection induced mitogen-activated protein kinase activation, but did not elicit NFκB activation, thereby affecting the production of pro-inflammatory cytokines. The Shimen strain infection resulted in a significant activation of IFN regulatory factor IRF7 and suppression of IRF3. These data provided clues for understanding the effect of CSFV infection on the TLR-mediated innate immune response and associated pathological changes.

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that induces persistent diarrhoea in swine, resulting in severe economic losses in swine-producing countries. Insights into the interplay between PEDV infection and the innate immune system are necessary for understanding the associated mechanism of pathogenesis. The transcription factor NF-κB plays an important role in regulating host immune responses. Here, we elucidated for the first time to our knowledge the potential mechanism of PEDV-mediated NF-κB activation in porcine small intestinal epithelial cells (IECs). During PEDV infection, NF-κB p65 was found to translocate from the cytoplasm to the nucleus, and PEDV-dependent NF-κB activity was associated with viral dose and active replication. Using small interfering RNAs to screen different mRNA components of the Toll-like receptor (TLR) or RIG-I-like receptor signalling pathways, we demonstrated that TLR2, TLR3 and TLR9 contribute to NF-κB activation in response to PEDV infection, but not RIG-I. By screening PEDV structural proteins for their ability to induce NF-κB activities, we found that PEDV nucleocapsid protein (N) could activate NF-κB and that the central region of N was essential for NF-κB activation. Furthermore, TLR2 was involved in PEDV N-induced NF-κB activation in IECs. Collectively, these findings provide new avenues of investigation into the molecular mechanisms of NF-κB activation induced by PEDV infection.

Rotavirus is a leading cause of severe gastroenteritis in infants worldwide. Rotavirus nonstructural protein 1 (NSP1) is a virulence factor that inhibits innate host immune responses. NSP1 from some rotaviruses targets host interferon response factors (IRFs), leading to inhibition of type I interferon expression. A few rotaviruses encode an NSP1 that inhibits the NF-κB pathway by targeting β-TrCP, a protein required for IκB degradation and NF-κB activation. Available evidence suggests that these NSP1 properties involve proteosomal degradation of target proteins. We show here that NSP1 from several human rotaviruses and porcine rotavirus CRW-8 inhibits the NF-κB pathway, but cannot degrade IRF3. Furthermore, β-TrCP levels were much reduced in cells infected with these rotaviruses. This provides strong evidence that β-TrCP degradation is required for NF-κB pathway inhibition by NSP1 and demonstrates the relevance of β-TrCP degradation to rotavirus infection. C-terminal regions of NSP1, including a serine-containing motif resembling the β-TrCP recognition motif of IκB, were required for NF-κB inhibition. CRW-8 infection of HT-29 intestinal epithelial cells induced significant levels of IFN-β and CCL5 but not IL-8. This contrasts with monkey rotavirus SA11-4F, whose NSP1 inhibits IRF3 but not NF-κB. Substantial amounts of IL-8 but not IFN-β or CCL5 were secreted from HT-29 cells infected with SA11-4F. Our results show that human rotaviruses commonly inhibit the NF-κB pathway by degrading β-TrCP and thus stabilizing IκB. They suggest that NSP1 plays an important role during human rotavirus infection by inhibiting the expression of NF-κB-dependent cytokines, such as IL-8.

Five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified in the genus Orthoreovirus. Their genomes each consist of 10 dsRNA segments. A novel orthoreovirus was isolated from the haemorrhagic intestine of a dead brown-eared bulbul (Hypsipetes amaurotis) in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed non-enveloped capsids of ~70 nm diameter, which were characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and σC, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV, but was located on a phylogenetic branch different from that of ARV and NBV. The virus had the closest phylogenetic relationship to two reovirus strains: SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The 10 RNA segments had a 3′ pentanucleotide sequence (UCAUC-3′) conserved amongst all members of the genus Orthoreovirus, and a unique 5′ terminal heptasequence (5′-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggested that the novel virus might form a new species with the two strains in the genus Orthoreovirus.

In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus–host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.

Grass carp reovirus (GCRV) is a member of the genus Aquareovirus in the family Reoviridae, and contains five core proteins (VP1–VP4 and VP6) and two outer-capsid proteins (VP5 and VP7) in its particle. Previous studies have revealed that the outer-capsid proteins of reovirus are responsible for initiating infection, but the mechanism is poorly understood. Using baculovirus-expressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV could be inhibited by treating cells with the weak base NH4Cl. In addition, recoated particles carrying an Asn→Ala substitution at residue 42 of VP5 (VP5N42A/VP7 R-GCRV) were no longer infectious. These results provide strong evidence that autocleavage of VP5 is critical for aquareovirus to initiate efficient infection.

Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically. During 2009, 10 G10P[8] rotavirus strains were detected in the stools of children hospitalized with acute diarrhoea in several paediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P[8] strains analysed revealed nucleotide identities ranging from 94 to 99 %. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Amongst the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains. A hydrophobic cluster analysis (HCA) of the VP7 protein including aa 20–298 was performed for the G10 Italian sequences in comparison with the major human group A rotavirus G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.

Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date, the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrated that extracellular treatment of BHK-21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, was sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment were required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2/VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicated that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, was required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study showed that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways were induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells did not diminish AHSV4 replication, but reduced the release and dissemination of progeny viral particles. Taken together, the data indicated that uncoating of AHSV virions was required for apoptosis induction, and that apoptosis enhanced virus spread and release.

Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70 %. To explore host/pathogen interactions, a next-generation sequencing approach using lymphoid organ tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 bp at the 5′ terminus and 23 nt at the 3′ terminus as compared with the original description of the IMNV genome (7561 nt). Northern blot and reverse transcription-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 5′ RACE, an additional 4 nt were discovered; 3′ RACE confirmed the presence of 22 bp rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. dsRNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 5′ end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5′ UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger targeting a 381 bp sequence in ORF1 improved survival to 82.2 % as compared with 2.2 % survival in positive control animals. These studies revealed the importance of the new genome sections to produce high-titre infection, and associated disease and mortality, in infected shrimp.

The oldest porcine circovirus type 2 (PCV2) sequence dates back to 1962 and is among several hundreds of publicly available PCV2 sequences. Despite this resource, few studies have investigated the global genetic diversity of PCV2. To evaluate the phylogenetic relationship of PCV2 strains, 1680 PCV2 open reading frame 2 (ORF2) sequences were compared and analysed by methods of neighbour-joining, maximum-likelihood, Bayesian inference and network analysis. Four distinct clades were consistently identified and included PCV2a, PCV2b, PCV2c and PCV2d; the p-distance between PCV2d and PCV2b was 0.055±0.008, larger than the PCV2 genotype-definition cut-off of 0.035, supporting PCV2d as an independent genotype. Among the 1680 sequences, 278–285 (16.5–17 %) were classified as PCV2a, 1007–1058 (59.9–63 %) as PCV2b, three (0.2 %) as PCV2c and 322–323 (19.2 %) as PCV2d, with the remaining 12–78 sequences (0.7–4.6 %) classified as intermediate clades or strains by the various methods. Classification of strains to genotypes differed based on the number of sequences used for the analysis, indicating that sample size is important when determining classification and assessing PCV2 trends and shifts. PCV2d was initially identified in 1999 in samples collected in Switzerland, now appears to be widespread in China and has been present in North America since 2012. During 2012–2013, 37 % of all investigated PCV2 sequences from US pigs were classified as PCV2d and overall data analysis suggests an ongoing genotype shift from PCV2b towards PCV2d. The present analyses indicate that PCV2d emerged approximately 20 years ago.

Persistent infection with oncogenic human papillomavirus (HPV) is a prerequisite for cervical disease development, yet data regarding the host immune response to infection at the genotype level are quite limited. We created pseudoviruses bearing the major (L1) and minor (L2) capsid proteins and L1 virus-like particles representing the reference sequence and a consensus of 34 European sequences of HPV51. Despite the formation of similarly sized particles, motifs in the reference L1 and L2 genes had a profound impact on the immunogenicity, antigenicity and infectivity of these antigens. The antibody status of women exhibiting low-grade disease was similar between HPV16 and the consensus HPV51, but both demonstrated discrepancies between binding and neutralizing antibody responses. These data support the use of pseudoviruses as the preferred target antigen in studies of natural HPV infection and the need to consider variation in both the L1 and L2 proteins for the appropriate presentation of antibody epitopes.

Hepatitis B virus (HBV) infection is associated with a broad spectrum of clinical manifestations, including cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress and subsequent oxidative stress have been implicated in liver carcinogenesis and disease progression with chronic inflammation. In our previous study, several mutations in the precore/core region of HBV genotype C were identified from 70 Korean chronic patients, and the mutations were associated with HCC and/or HBV e antigen serostatus. Here, we found that the naturally occurring mutations P5T/H/L of the HBV core antigen induced ER stress. The upregulation of ER stress resulted in higher reactive oxygen species production, intracellular calcium concentration, inflammatory cytokines as well as surface antigen production and apoptosis of cells. This study suggested that these mutations may contribute to the progression of liver disease in chronic patients.

Human cytomegalovirus (CMV) is the most common infectious cause of congenital birth defects in developed countries. Studies of infected amniotic fluid and placentae show CMV infection leads to a pro-inflammatory shift in cytokine profiles with implications for pathogenesis of foetal disease. ELISA, immunofluorescence and real-time-PCR assays were used to investigate CCL2 (monocyte chemotactic protein-1) and TNF-α changes following CMV infection of human fibroblasts, as well as following transient expression of CMV gene products in HeLa cells. Infection of human fibroblasts with CMV AD169 resulted in increased cytoplasmic and extracellular expression of CCL2 during early stages of infection, followed by marked downregulation of the chemokine at late times. Induction of CCL2 was not observed with CMV clinical strain Merlin, consistent with the postulated immune-evasion potential of this genetically intact WT strain. Comparison between live and UV-irradiated virus infections showed that changes in CCL2 levels were a direct response to active CMV replication. There were no significant changes in TNF-α expression during a parallel time-course of CMV infection. In transient transfection assays, overexpression of CMV tegument protein pp71 resulted in intracellular and extracellular upregulation of CCL2 protein. mRNA analysis showed that pp71-induced elevation in CCL2 was mediated through transcriptional upregulation. The data showed that CMV-induced upregulation of CCL2 during early stages of infection was mediated, at least in part, by stimulation of viral pp71, which may contribute to viral pathogenesis through enhanced virus dissemination.

Lymphoma is the most common haematopoietic malignancy in dogs, but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein–Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it was suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41 % of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridization or PCR, and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.

The genome of the rat cytomegalovirus (RCMV) English isolate (MuHV-8) differs significantly from the RCMV Maastricht isolate (MuHV-2) and other cytomegaloviruses (CMVs) in its size, base composition and genomic content. Analysis of the RCMV-Berlin isolate, MuHV-8, revealed that the two MuHV-8 isolates are highly similar in genome size and content, indicating that the smaller genome size (202 946 bp) compared to other known CMVs was not the result of an accidental deletion during passage in tissue culture. Surprisingly, the proteins encoded in MuHV-8 shared more overall similarity with their orthologues from mouse CMV (MuHV-1) compared to their orthologues in rat CMV (MuHV-2). Phylogenetic analyses of conserved viral genes showed that the two MuHV-8 isolates are from the same species and represent a unique clade that is distinct from other rodent CMVs.

The open reading frame 45 (ORF45) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early phosphorylated tegument protein critical for viral escape from host immune surveillance. Its expression is upregulated by the viral replication and transcription activator (RTA), a key protein that controls the switch from latency to lytic replication. We report here that ORF45 expression was not only upregulated by RTA, but ORF45 could also be degraded by RTA in a proteasome-dependent manner. The ORF45 was activated by RTA via activation of the ORF45 promoter, and the promoter region from nt 69 271 to nt 69 026 was involved. In chronic KSHV infected TRE-BCBL-1 RTA cells, the endogenous ORF45 protein increased dramatically after the induction of RTA expression, but then decreased rapidly after 8 h post-induction. Our study suggests that RTA might control the kinetics of viral replication through fine-tuning of the level of ORF45 and other viral/host proteins.

HIV prevalence has decreased in Uganda since the 1990s, but remains substantial within high-risk groups. Here, we reconstruct the history and spread of HIV subtypes A1 and D in Uganda and explore the transmission dynamics in high-risk populations. We analysed HIV pol sequences from female sex workers in Kampala (n = 42), Lake Victoria fisher-folk (n = 46) and a rural clinical cohort (n = 74), together with publicly available sequences from adjacent regions in Uganda (n = 412) and newly generated sequences from samples taken in Kampala in 1986 (n = 12). Of the sequences from the three Ugandan populations, 60 (37.1 %) were classified as subtype D, 54 (33.3 %) as subtype A1, 31 (19.1 %) as A1/D recombinants, six (3.7 %) as subtype C, one (0.6 %) as subtype G and 10 (6.2 %) as other recombinants. Among the A1/D recombinants we identified a new candidate circulating recombinant form. Phylodynamic and phylogeographic analyses using BEAST indicated that the Ugandan epidemics originated in 1960 (1950–1968) for subtype A1 and 1973 (1970–1977) for D, in rural south-western Uganda with subsequent spread to Kampala. They also showed extensive interconnection with adjacent countries. The sequence analysis shows both epidemics grew exponentially during the 1970s–1980s and decreased from 1992, which agrees with HIV prevalence reports in Uganda. Inclusion of sequences from the 1980s indicated the origin of both epidemics was more recent than expected and substantially narrowed the confidence intervals in comparison to previous estimates. We identified three transmission clusters and ten pairs, none of them including patients from different populations, suggesting active transmission within a structured transmission network.

All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.