The Emergence of Zika Virus as a Global Health Security Threat: A Review and a Consensus Statement of the INDUSEM Joint working Group (JWG).

The Zika virus (ZIKV), first discovered in 1947, has emerged as a global public health threat over the last decade, with the accelerated geographic spread of the virus noted during the last 5 years. The World Health Organization (WHO) predicts that millions of cases of ZIKV are likely to occur in the Americas during the next 12 months. These projections, in conjunction with suspected Zika-associated increase in newborn microcephaly cases, prompted WHO to declare public health emergency of international concern. ZIKV-associated illness is characterized by an incubation period of 3-12 days. Most patients remain asymptomatic (i.e., ~80%) after contracting the virus. When symptomatic, clinical presentation is usually mild and consists of a self-limiting febrile illness that lasts approximately 2-7 days. Among common clinical manifestations are fever, arthralgia, conjunctivitis, myalgia, headache, and maculopapular rash. Hospitalization and complication rates are low, with fatalities being extremely rare. Newborn microcephaly, the most devastating and insidious complication associated with the ZIKV, has been described in the offspring of women who became infected while pregnant. Much remains to be elucidated about the timing of ZIKV infection in the context of the temporal progression of pregnancy, the corresponding in utero fetal development stage(s), and the risk of microcephaly. Without further knowledge of the pathophysiology involved, the true risk of ZIKV to the unborn remains difficult to quantify and remediate. Accurate, portable, and inexpensive point-of-care testing is required to better identify cases and manage the current and future outbreaks of ZIKV, including optimization of preventive approaches and the identification of more effective risk reduction strategies. In addition, much more work needs to be done to produce an effective vaccine. Given the rapid geographic spread of ZIKV in recent years, a coordinated local, regional, and global effort is needed to generate sufficient resources and political traction to effectively halt and contain further expansion of the current outbreak.

A structural analysis of M protein in coronavirus assembly and morphology.

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

A new cistron in the murine hepatitis virus replicase gene.

We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

Fate of minus-strand templates and replication complexes produced by a p23-cleavage-defective mutant of Sindbis virus.

SIN2V is an engineered mutant Sindbis virus (SIN) that is unable to process the P23 cleavage site in polyproteins P123 and P1234 that are translated from the genome after its entry into cells. Unlike wild-type (wt) SIN, it caused minus strands to be made continuously and replication-transcription complex (RTC) activity to be unstable (R. Gorchakov, E. Frolova, S. Sawicki, S. Atasheva, D. Sawicki, and I. Frolov, J. Virol. 82:6218-6231, 2008). We examined further the effects of P23 on SIN RNA replication and RTC activity. Continuous minus-strand synthesis by SIN2V produced 250% of wt levels of minus strands but accumulated only 110% of wt levels (0.39 pg, or 2.7 x 10(4) molecules of double-stranded RNA per cell). Because SIN2V-infected cells accumulated only 40% of the minus strands that were made, cells must possess some process to limit RTC accumulation. The loss of activity by SIN2V RTC after translation was inhibited was stochastic and not due to their inherent instability, based on finding that activity was lost without the degradation of the minus-strand templates. In addition to their normal functions, P23 RTCs exhibited the novel phenotype of being unable to switch from making less to making more genomes than subgenomic 26S mRNA at late times during infections. Our results lend credence to the hypothesis that nsP2 (and possibly nsP3) possesses functions other than those needed solely for RTC activity and that it may also act with the host to regulate minus-strand synthesis and the stability of the RTC.

Molecular defects caused by temperature-sensitive mutations in Semliki Forest virus nsP1.

Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

Heparan sulfate is a binding molecule but not a receptor for CEACAM1-independent infection of murine coronavirus.

A highly neurovirulent mouse hepatitis virus (MHV) JHMV strain (wt) with receptor (MHVR)-independent infection activity and its low-virulent mutant srr7 without such activity were found to attach to MHVR-negative, non-permissive BHK cells. To identify the molecule that interacts with JHMV, we focused on heparan sulfate (HS) since it works as a receptor of a mutant MHV-rec1 that infects in an MHVR-independent fashion. The present study indicates that HS interacts with both wt JHMV and srr7 but it does not function as an entry receptor as it apparently does for MHV-rec1. Furthermore, HS failed to serve as an entry receptor in the MHVR-independent infection of wt JHMV, indicating that HS is not a host factor that wt JHMV utilizes in an MHVR-independent infection.

Role for nsP2 proteins in the cessation of alphavirus minus-strand synthesis by host cells.

In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and approximately 10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

Functional and genetic analysis of coronavirus replicase-transcriptase proteins.

The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (non-structural proteins nsp1-nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12-nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II-VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the non-permissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.

Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo.

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells.

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.

Functional analysis of nsP3 phosphoprotein mutants of Sindbis virus.

Alphavirus nsP3 phosphoprotein is essential for virus replication and functions initially within polyprotein P123 or P23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsP3 likely functions also in plus-strand synthesis. We report the identification of a second nsP3 mutant from among the A complementation group of Sindbis virus (SIN) heat-resistant strain, ts RNA-negative mutants. The ts138 mutant possessed a change of G4303 to C, predicting an Ala68-to-Gly alteration that altered a conserved His-Ala-Val tripeptide in the ancient (pre-eukaryotic), "X" or histone 2A phosphoesterase-like macrodomain that in SIN encompasses nsP3 residues 1 to 161 and whose role is unknown. We undertook comparative analysis of three nsP3 N-terminal region mutants and observed (i) that nsP3 and nsP2 functioned initially as a single unit as deduced from complementation analysis and in agreement with our previous studies, (ii) that the degree of phosphorylation varied among the nsP3 mutants, and (iii) that reduced phosphorylation of nsP3 correlated with reduced minus-strand synthesis. The most striking phenotype was exhibited by ts4 (Ala268 to Val), which after shift to 40 degrees C made significantly underphosphorylated P23/nsP3 and lost selectively the ability to make minus strands. After shift to 40 degrees C, mutant ts7 (Phe312 to Ser) made phosphorylated P23/nsP3 and minus strands but failed to increase plus-strand synthesis. Macrodomain mutant ts138 was intermediate, making at 40 degrees C partially phosphorylated P23/nsP3 and reduced amounts of minus strands. The mutants were able to assemble their nsPs at 40 degrees C into complexes that were membrane associated. Our analyses argue that P23/P123 phosphorylation is affected by macrodomain and Ala268 region sequences and in turn affects the efficient transcription of the alphavirus genome.

Alphavirus minus-strand synthesis and persistence in mouse embryo fibroblasts derived from mice lacking RNase L and protein kinase R.

We report our studies to probe the possible role of the host response to double-stranded RNA in cessation of alphavirus minus-strand synthesis. Mouse embryo fibroblasts (MEF) from Mx1-deficient mice that also lack either the protein kinase R (PKR) or the latent RNase L or both PKR and RNase L were screened. In RNase L-deficient but not wild-type or PKR-deficient MEF, there was continuous synthesis of minus-strand templates and the formation of new replication complexes producing viral plus strands. Inhibiting translation caused minus-strand synthesis to stop and a loss of transcription activity of the mature replication complexes. This turnover of replication complexes that were stable in cells containing RNase L suggested that RNase L plays some role, albeit possibly indirect, in the formation of stable replication complexes during alphavirus infection. In addition, confluent monolayers of RNase L-deficient murine cells readily established persistent infections and were not killed. This phenotype is contrary to what has been observed for infection in vertebrate cells with a presumably functional RNase L gene and more resembled alphavirus replication in Aedes mosquito cells, in which the activity of replication complexes making plus stands was also found to decay with inhibition of translation.

Alphavirus minus-strand RNA synthesis: identification of a role for Arg183 of the nsP4 polymerase.

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40 degrees C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.

Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant.

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40 degrees C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40 degrees C. In Aedes albopictus cells at 34.5 degrees C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40 degrees C ( approximately 140%) and in Aedes cells at 34.5 degrees C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.

The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.