Current status of diagnostic methods for henipavirus.

Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of emerging transboundary animal disease in pigs and horses. They also cause fatal disease in humans. NiV has a case fatality rate of 40 - 100%. In the initial NiV outbreak in Malaysia in 1999, about 1.1 million pigs had to be culled. The economic impact was estimated to be approximately US$450 million. Worldwide, HeV has caused more than 60 deaths in horses with 7 human cases and 4 deaths. Since the initial outbreak, HeV spillovers from Pteropus bats to horses and humans continue. This article presents a brief review on the currently available diagnostic methods for henipavirus infections, including advances achieved since the initial outbreak, and a gap analysis of areas needing improvement.

Nipah virus: a recently emergent deadly paramyxovirus.

A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

Molecular biology of Hendra and Nipah viruses.

The structure and genetic organization of Hendra and Nipah viruses places them in the subfamily Paramyxovirinae. However, low homology with other subfamily members and several novel biological and molecular features such as genome length and F(0 )cleavage site suggest classification in a new genus within the Paramyxovirinae.

New genetic group of measles virus isolated in the People's Republic of China.

Genetic and antigenic characterization of 14 wild-type measles viruses isolated from four provinces in the People's Republic of China during 1993 and 1994 was conducted. Sequence analyses of the hemagglutinin (H) and nucleoprotein (N) genes indicated that 13 of the 14 Chinese viruses comprised a previously undescribed genetic group. Viruses from this unique group were the most genetically diverse measles viruses described, so far. The Chinese viruses differed from other wild-type viruses by as much as 6.9% in the H gene and 7.0% in the N gene at the nucleotide level. One of the 14 viruses was a member of the same genetic group that contains the Edmonston strain. Antigenic analysis using monoclonal antibodies to the H protein did not detect significant differences in binding patterns between the Chinese viruses and other wild-type measles viruses. In addition, representative viruses from the unique Chinese group were neutralized by both human post-vaccination antiserum and mouse antiserum against the H protein of the Edmonston vaccine virus. Viruses closely related to these Chinese viruses were also associated with importations of measles into the United States during 1997 from Vietnam and Hong Kong suggesting that viruses from this new genetic group continue to circulate in China and possibly other parts of Asia.

A single nucleotide substitution in the 5'-untranslated region of the vaccinia N2L gene is responsible for both alpha-amanitin-resistant and temperature-sensitive phenotypes.

The locus responsible for encoding resistance to alpha-amanitin was previously mapped to the vaccinia virus (VV) HindIII N fragment by using cloned wild-type VV DNA fragments to rescue the ability of an alpha-amanitin-resistant/temperature-sensitive VV mutant (alpha rts7) to replicate under nonpermissive conditions. DNA sequencing and transcriptional analyses of this region identified two leftward-reading open reading frames (ORFs), N2L and M1L, as candidates to encode the protein responsible for eliciting both phenotypes. In the present study, high-resolution marker rescue mapping and genomic sequencing techniques have been applied to identify the nature of the mutation within the HindIII N region of the alpha rts7 genome. Interestingly, a single G to T transversion mutation was noted at position -10 relative to the initiator ATG of the N2L ORF. Since transcription of the N2L gene starts at position -12/-13, this places the alpha rts7 mutation within the 5'-untranslated leader of the N2L transcript expressed early in infection and suggests that the transcriptional efficiency, mRNA stability, or translational efficiency must be altered in the mutant RNA. These results identify the N2L ORF as the gene responsible for conferring resistance to alpha-amanitin in the alpha rts7 mutant and suggest that the N2L gene product is the viral function that interacts with the host cell nucleus during VV infection.

Anchoring a vaccinia virus promoter in the nucleus prevents its trans-activation by viral infection.

The vaccinia virus 7.5 kDa constitutive promoter, when fused to a reporter gene and recombined into the genome of L cells, is not activatable upon subsequent infection with vaccinia virus. However, the same promoter is actively transcribed during transient cytoplasmic transfection procedures or within the context of the viral genome. This suggests that the intact vaccinia transcriptional machinery either does not enter the nucleus or, if it does, is unable to interact with cellular chromatin.

Nucleotide sequence and molecular genetic analysis of the vaccinia virus HindIII N/M region encoding the genes responsible for resistance to alpha-amanitin.

The genomic location of the gene(s) which provides vaccinia virus (VV) alpha-amanitin-resistant mutants with a drug-resistant phenotype have been mapped to the HindIII N/M region of the genome by the use of marker rescue techniques [E. C. Villarreal and D. E. Hruby (1986) J. Virol. 57, 65-70]. Nucleotide sequencing of a 2356-bp HindIII-Sau3A fragment of the vaccinia virus genome encompassing this region reveals the presence of two complete leftward-reading open reading frames (ORFs, N2 and M1) and two incomplete ORFs (N1 and M2). By computer analysis the N2 and M1 ORFs would be predicted to encode soluble VV polypeptides with molecular weights of approximately 20 and 48 kDa, respectively. The N2 and M1 ORFs have extremely A-T-rich 5'-proximal sequences, consistent with previous data regarding the location and A-T-richness of viral early promoters. Likewise, the consensus signal believed to be involved in terminating VV early gene transcription, TTTTTNT, was evident at the 3'-boundary of both the N2 and M1 ORFs suggesting that these genes may be VV early genes. The in vivo transcriptional activity, orientation, and limits of these putative transcriptional units were investigated by Northern blot, nuclease S1, and primer extension analysis. Both N2- and M1-specific transcripts were detected in the cytoplasm of VV-infected cells, suggesting that these loci are bonafide viral genes. Time-course nuclease S1 experiments revealed that the N2 gene was transcribed exclusively prior to VV DNA replication. In contrast, the M1 gene was transcribed throughout infection, although different start sites were used at early versus late times postinfection. These results are discussed in relation to the drug-resistant phenotype and future experiments to identify the viral gene product responsible.

Antigenic analysis of current wild type and vaccine strains of measles virus.

The antigenic properties of vaccine and wild type strains of measles virus were compared. Serum specimens from vaccinated persons, persons infected during the prevaccine era, or mice experimentally vaccinated with the hemagglutinin (H) protein from vaccine virus neutralized vaccine virus and a wild type measles virus from 1989 equally well. In contrast, serum specimens from patients with recent measles virus infection and mice experimentally vaccinated with the H protein from the wild type virus from 1989 neutralized wild type virus with titers 4-8 times higher than those to vaccine virus. Several H protein-specific monoclonal antibodies could differentially recognize vaccine or wild type virus. These data show that the H proteins of the recent wild type viruses contain both conserved and new or modified antigenic determinants and are consistent with previous studies that described genetic drift in the H proteins of recent wild type viruses.

Further analyses of the orthopoxviruses volepox virus and raccoon poxvirus.

Volepox virus (VPX) from skin lesions on a vole and a piñon mouse caught in California and raccoon poxvirus (RCN) from raccoons trapped in Maryland were examined to begin elucidating their relationship to other orthopoxviruses, most of which are not known to be indigenous to the Americas. VPX and RCN produced pinpoint, nonhemorrhagic pocks on chick embryo chorioallantoic membranes. In cell cultures both viruses produced 1-mm diameter, irregular plaques, A-type inclusions (ATIs), and despite production of hemagglutinin, both viruses caused syncytia formation. Considerable cross-hybridization was seen between VPX and RCN DNA and the DNAs of other orthopoxviruses; however, HindIII cleavage site maps showed marked central and terminal region differences between VPX (222.8 kbp) and RCN (224.8 kbp) DNA and mapped DNAs of other orthopoxviruses. Cognate DNAs of the ATI 160-kDa protein and 38-kDa serine protease inhibitor homologue of cowpox virus (CPV) and the 14-kDa fusion protein of vaccinia virus (VAC) were present within the right end of VPX and RCN DNA, matching their location in CPV and VAC. VPX and RCN, respectively, expressed a 150- and a 155-kDa ATI major protein and a 20- and an 18-kDa fusion protein. Low stringency annealing suggested that cognate DNAs for the VAC growth factor and the alpha-amanitin target protein were present within the left end of VPX and RCN DNA, matching their location in VAC. Terminal tandem repeat sequences of VAC and RCN did not cross-hybridize with each other or with VPX DNA end fragments. Together, the data suggested that VPX and RCN are phylogenetically rather distant from orthopoxviruses not indigenous to the Americas, although genetic information is arranged as in other examined orthopoxviruses.

Molecular characterization of the polymerase gene and genomic termini of Nipah virus.

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

Evaluation of recombinant vaccinia virus--measles vaccines in infant rhesus macaques with preexisting measles antibody.

Immunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication-defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants.

Molecular characterization of Nipah virus, a newly emergent paramyxovirus.

Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.