- Volume 96, Issue 3, 2015

Coronaviruses (CoVs) have been studied for over 60 years, but have only recently gained notoriety as deadly human pathogens with the emergence of severe respiratory syndrome CoV and Middle East respiratory syndrome virus. The rapid emergence of these viruses has demonstrated the need for good models to study severe CoV respiratory infection and pathogenesis. There are, currently, different methods and models for the study of CoV disease. The available genetic methods for the study and evaluation of CoV genetics are reviewed here. There are several animal models, both mouse and alternative animals, for the study of severe CoV respiratory disease that have been examined, each with different pros and cons relative to the actual pathogenesis of the disease in humans. A current limitation of these models is that no animal model perfectly recapitulates the disease seen in humans. Through the review and analysis of the available disease models, investigators can employ the most appropriate available model to study various aspects of CoV pathogenesis and evaluate possible antiviral treatments that may potentially be successful in future treatment and prevention of severe CoV respiratory infections.

Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3′ end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.

Crimean–Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus of the family Bunyaviridae, that can cause severe haemorrhagic fever in humans, with mortality rates above 30 %. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative-sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N protein) and the medium segment (M) encodes the envelope proteins. The N protein of bunyaviruses binds genomic RNA, forming the viral ribonucleoprotein (RNP) complex. The L protein interacts with these RNP structures, allowing the initiation of viral replication. The N protein also interacts with actin, although the regions and specific residues involved in these interactions have not yet been described. Here, by means of immunoprecipitation and immunofluorescence assays, we identified the regions within the CCHFV N protein implicated in homo-oligomerization and actin binding. We describe the interaction of the N protein with the CCHFV L protein, and identify the N- and C-terminal regions within the L protein that might be necessary for the formation of these N–L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication.

Pathogenesis of viral haemorrhagic fevers is associated with alteration of vascular barrier function and haemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV). In this study, we demonstrated that CCHFV infection suppresses cleavage of poly(ADP-ribose) polymerase (PARP), triggered by staurosporine early post-infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid late post-infection, suggesting activation of extrinsic apoptotic signalling. Consistently, supernatant from cells stimulated late post-infection was found to induce PARP cleavage, most probably through the TNF-α death receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascades. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late stages post-infection induces host cellular pro-apoptotic molecules through the death receptor pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent throughout the world and has caused major economic losses to the pig industry. Arterivirus non-structural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication. PRRSV nsp10, however, has not yet been well characterized. In this study, a series of nsp10 mutants were constructed and analysed for functional sites of different enzymic activities. We found that nsp10 could bind both ssDNA and dsDNA, and this binding activity could be inactivated by mutations at Cys25 and His32. These two mutations also abolished unwinding activity without affecting ATPase activity. In addition, substitution of Ala227 by Ser eliminated helicase activity, whilst substitution by Val enhanced unwinding activity. Taken together, our results showed that Cys25 and His32 in PRRSV nsp10 were critical for nucleic acid binding and unwinding, and that Ala227 played an important role in helicase activity.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 βE-βF loop, VP1 C-terminus and VP3 β-B knob) surrounding the fivefold symmetry axis (VP1 βF-βG loop) and at other discrete sites on the capsid (VP3 βG-βH loop, VP1 C-terminus, VP2 βC strand and VP1 βG-βH loop). A lysine insertion in the VP1 βF-βG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 βG-βH loop (−3, −2 and +2 ‘RGD’ positions) or in the structural element known to be juxtaposed against that loop (VP1 βB-βC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the ‘RGD’ motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5′ end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3′ end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

Pleconaril is a capsid inhibitor used previously to treat enterovirus infections. A pleconaril-resistant echovirus 11 (E11) strain was identified before pleconaril treatment was given in an immunocompromised patient. The patient was also treated with intravenous Ig (IVIg) for a long period but remained unresponsive. The pleconaril-resistant strains could not be neutralized in vitro, confirming IVIg treatment failure. To identify the basis of pleconaril resistance, genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M and I188L) in the pleconaril-resistant viruses were found in the pocket region of VP1. Modelling suggested that V119M could confer resistance, most probably due to the protruding sulfate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterized by a different substitution (I183M), resistance was suggested to also result from a similar mechanism, i.e. due to a protruding sulfate side chain of methionine. Our results showed that resistant strains that arise in vivo display different markers from those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIg treatment failure, we predict that one of these factors could be immune related. Thus, both IVIg and capsid inhibitors target the viral capsid and can induce mutations that can be cross-reactive, enabling escape from both IVIg and the drug. This could limit treatment options and should be investigated further.

The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

Rotavirus (RV) A is a very common cause of acute diarrhoea in infants and young children worldwide. Most human strains are classified into two major Wa-like and DS-1-like genotype constellations, whilst a minor third strain, AU-1, was described in 1989 among human RV isolates from Japan. AU-1 demonstrates a high degree of homology to a feline RV, FRV-1, which suggests interspecies transmission of feline RV. However, there has been no subsequent report of RVs possessing the AU-1 genotype throughout all 11 genes of the genome. Between March 1997 and December 1999, 157 RV-positive stool samples were collected from Brazilian children, and 16 of the RVs (10.2 %) were P[9] genotype. We analysed eight strains by almost full-genome sequencing. These eight strains were divided into two groups: five AU-1-like and three Wa-like strains. Four of the five AU-1-like strains had the AU-1-like genotype constellation throughout the 11 genes. The remaining AU-1-like strain was considered to be a reassortant strain comprosed of nine, two and one genes from the AU-1-like, Wa-like and G9 strains, respectively. The three Wa-like strains were considered to be reassortants comprising seven to eight genes and three to four genes from Wa-like and non-Wa-like strains, respectively. This report of human G3P[9] RV strains possessing the AU-1 genotype constellation throughout all genes demonstrates the stability and infectivity of the AU-1-like strain with its original genotype over distance and time.

In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an ‘AT trap’, an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly(A : T) tract or variants of the SV40 WT sequence all restored infectious virus. In a transfection of the AT trap without a suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.

Human papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through faeces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analysed 103 faecal specimens collected from hospitalized patients with diarrhoea using validated primers able to detect α, β and γ HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the faecal samples. Thirteen samples (12.6 %) were positive for nine genotypes belonging to the α and β genera: HPV32 (LR, α1), HPV39 (HR, α7), HPV44 (LR, α10), HPV8 (β1), HPV9, HPV23, HPV37, HPV38 and HPV120 (β2). Two putative novel genotypes of the β genus, species 1 and 2, were also detected. The tissue(s) of origin is unknown, since faeces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in faecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a consequence of shedding in the faeces. This highlights the need for further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.

Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal-truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx- nor FL-HBx-transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN) at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx-transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that, statistically, FL-HBx mice developed more liver lesions with a larger surface area than control mice. Furthermore, during DEN-induced initiation of HCC, Ct-HBx- and FL-HBx-transgenic mice showed higher expression of IL-6, TNF-α and IL-1β transcripts, activation of STAT3, ERK and JNK proteins and an increase in cell apoptosis. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than does FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein–Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

Marek’s disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXΔmiR-M2, GXΔmiR-M3, GXΔmiR-M5, GXΔmiR-M9 and GXΔmiR-M12 was reduced from 100 % to 18 %, 30 %, 48 %, 24 % and 14 %, coupled with gross tumour incidence reduction from 28 % to 8 %, 4 %, 12 %, 8 % and 0 %, respectively. Our data confirm that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.

Mouse mammary tumour virus (MMTV) is a member of the genus Betaretrovirus, infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV from C3H mice [MMTV(C3H)], reported previously to be incapable of infecting human cells, could productively infect human cells. Using a recently described high-titre MMTV-based vector carrying MMTV(C3H) envelope protein (Env), we successfully transduced cells of human origin. Furthermore, WT MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. The infectivity of WT MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat inactivation, an inhibitor of reverse transcription (3′-azido-3′-deoxythymidine) and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic murine leukemia virus Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naïve human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells were related antigenically to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able not only to infect but also to replicate in cultured human breast cells.

Chronic hepatitis B (CHB) is treated with nucleos(t)ide analogues (NAs). The reverse transcriptase (RT) region in the hepatitis B virus (HBV) genome mutates to resist NA treatment, yet the RT mutations have not been well characterized. Furthermore, the HBV genotype might influence RT sequence evolution, NA resistance (NAr) mutation patterns and drug resistance development. We examined 42 NAr mutation sites in 169 untreated and 131 NA-treated CHB patient samples. Patients were identified with HBV-B and HBV-C genotype infections, with a higher prevalence and mutation frequency of HBV-C than HBV-B. Seventeen reported NAr mutation sites and 13 novel mutations were detected. NAr-related mutation prevalence was significantly higher in NA-treated versus untreated patients. Primary antiviral-resistant mutants only existed in NA-treated patients. Sequencing data revealed seven HBV-C-specific mutations and three HBV-B-specific mutations. In conclusion, NA treatment and HBV genotype might constitute the selection basis and promote NA-resistant HBV strain evolution under antiviral therapy.

Neutralizing antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here, we present studies where we examined the neutralization of HIV-luciferase pseudotypes bearing homologous and heterologous FIV envelope proteins (n = 278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n = 38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants, as well as recombinants, and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4+ T lymphocyte numbers, health status and survival times of the infected cats. Neutralization profiles varied significantly between FIV-infected cats and strong autologous neutralization, assessed using luciferase-based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms were likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralizing antibodies were evident in only a small proportion (13 %) of cats.