A novel hantavirus associated with an outbreak of fatal respiratory disease in the southwestern United States: evolutionary relationships to known hantaviruses.

Four Corners hantavirus (FCV) is the tentative name of the suspected etiologic agent of the newly identified hantavirus-associated respiratory distress syndrome (HARDS). The identification in HARDS patients of serum immunoglobulin M and immunoglobulin G antibodies that cross-reacted with Hantaan, Seoul, and Puumala virus antigens first suggested that FCV is a hantavirus. Limited nucleotide sequence data from the FCV glycoprotein-2 (G2) confirmed that FCV is a hantavirus and showed that it is most closely related to Prospect Hill and Puumala viruses. We have molecularly cloned approximately 95% of the sequences of the M and S segments of the FCV genome encoding the envelope glycoproteins and nucleocapsid protein N from the lungs of a patient with HARDS. The nucleotide sequence has been determined for 2,632 bases. The nucleotide sequence data show that FCV is a new member of the Puumala virus and Prospect Hill virus division of the hantavirus genus. Phylogenetic tree analyses indicate that the M and S segments have evolved in parallel. Therefore, the novel pathogenic activity of FCV is not likely to be the result of recent reassortment of segments from less pathogenic viruses.     

Cross-species transmission in the speciation of the currently known murinae-associated hantaviruses

To gain more insight into the phylogeny of Dabieshan virus (DBSV), carried by Niviventer confucianus and other Murinae-associated hantaviruses, genome sequences of novel variants of DBSV were recovered from Niviventer rats trapped in the mountainous areas of Wenzhou, China. Genetic analyses show that all known genetic variants of DBSV, including the ones identified in this study, are distinct from other Murinae-associated hantaviruses. DBSV variants show geographic clustering and high intraspecies diversity. The data suggest that DBSV is a distinct species in the genus Hantavirus. Interestingly, DBSV shows the highest sequence identity to Hantaan virus (HTNV), with a >7% difference in the sequences of the N, GPC, and L proteins, while N. confucianus is more closely related to Rattus norvegicus (the host of Seoul virus [SEOV]) than to Apodemus agrarius (the host of HTNV and Saaremaa virus [SAAV]). Further genetic analyses of all known Murinae-associated hantaviruses (both established and tentative species) show that many of them, including DBSV, may have originated from host switching. The estimation of evolutionary rates and divergence time supports the role of cross-species transmission in the evolution of Murinae-associated hantaviruses. The detection of positive selection suggests that genetic drift may contribute to the speciation of Murinae-associated hantaviruses and that adaptation has a role as well.     

Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle

A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.     

The genome organization of STLV-3 is similar to that of the AIDS virus except for a truncated transmembrane protein

Nucleotide sequence analysis of the 3' portion of the genome of simian T-lymphotropic virus type 3 from African green monkeys (STLV-3agm) reveals that it has the same general genome structure as the human immunodeficiency virus (HIV-1), the etiologic agent of AIDS. Short segments of strong amino acid homology and similar predicted protein structure characterize the tat and art/trs open reading frames (orf) of both viruses. Strong conservation of 3' orf and of another, cs-orf, for which no protein product has been identified suggests that they both encode proteins important to the life cycle of these viruses. The extracellular glycoproteins of STLV-3 and HIV-1 share a similar backbone structure and 50%-55% amino acid homology in constant domains of the HIV-1 protein. The most evident departure in structural organization is truncation of the transmembrane glycoproteins in two STLV-3agm clones and a biologically active, noncytopathic clone of HTLV-4.     

Analysis of hantavirus genetic diversity in Argentina: S segment-derived phylogeny

Nucleotide sequences were determined for the complete S genome segments of the six distinct hantavirus genotypes from Argentina and for two cell culture-isolated Andes virus strains from Chile. Phylogenetic analysis indicates that, although divergent from each other, all Argentinian hantavirus genotypes group together and form a novel phylogenetic clade with the Andes virus. The previously characterized South American hantaviruses Laguna Negra virus and Rio Mamore virus make up another clade that originates from the same ancestral node as the Argentinian/Chilean viruses. Within the clade of Argentinian/Chilean viruses, three subclades can be defined, although the branching order is somewhat obscure. These are made of (i) "Lechiguanas-like" virus genotypes, (ii) Maciel virus and Pergamino virus genotypes, and (iii) strains of the Andes virus. Two hantavirus genotypes from Brazil, Araraquara and Castello dos Sonhos, were found to group with Maciel virus and Andes virus, respectively. The nucleocapsid protein amino acid sequence variability among the members of the Argentinian/Chilean clade does not exceed 5.8%. It is especially low (3.5%) among oryzomyine species-associated virus genotypes, suggesting recent divergence from the common ancestor. Interestingly, the Maciel and Pergamino viruses fit well with the rest of the clade although their hosts are akodontine rodents. Taken together, these data suggest that under conditions in which potential hosts display a high level of genetic diversity and are sympatric, host switching may play a prominent role in establishing hantavirus genetic diversity. However, cospeciation still remains the dominant factor in the evolution of hantaviruses.     

A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana.

Genetic analysis of virus detected in autopsy tissues of a fatal hantavirus pulmonary syndrome-like case in Louisiana revealed the presence of a previously unrecognized hantavirus. Nucleotide sequence analysis of PCR fragments of the complete S and M segments of the virus amplified from RNA extracted from the tissues showed the virus to be novel, differing from the closest related hantavirus, Sin Nombre virus, by approximately 30%. Both genome segments were unique, and there was no evidence of genetic reassortment with previously characterized hantaviruses. The primary rodent reservoir of Sin Nombre virus, the deer mouse Peromyscus maniculatus, is absent from Louisiana. Thus, the virus detected in Louisiana, referred to here as Bayou virus, must possess a different rodent reservoir.     

在我国发现普马拉病毒新亚型

对我国东北地区捕获的157份棕背鼠平的肺组织进行普马拉病毒检测.其中免疫荧光检测出阳性标本1份,PCR检测出核苷酸阳性标本7份.对PCR产物进行测序后发现,其核苷酸的序列与报道的普马拉病毒核苷酸序列存在着差异.系统进化分析表明,我国发现的这株普马拉病毒,在进化树上形成了一个新的分支,为一新亚型.并且与在韩国和日本发现的普马拉病毒亲缘关系最为接近.     

The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.     

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3' and 5' ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.     

Genomic and copy-back 3'' termini in Sendai virus defective interfering RNA species

Direct sequencing of nine Sendai virus defective interfering RNA species revealed two kinds of 3'-terminal sequences. Six RNA species had 3' termini identical to the virus genome (negative strand), confirming that internal deletions are a frequent cause of Sendai virus defectiveness. The other three RNA species had 3'-terminal sequences identical to that described as the complement of the 5' terminus of the virus genome (R. A. Lazzarini, J. D. Keene, and M. Schubert, Cell 26:145-154, 1981), indicating that they are of the copy-back type. Extensive homology between these two types of 3' sequences evidently accounts for the ability of the copy-back sequence to function as an initiation signal for viral RNA replication. There may not be a selective advantage of one type of terminus over the other, since one defective interfering strain possessed two RNA species, one of which had the genomic 3' terminus and the other copy-back type.     

汉坦病毒S基因的克隆及体外转录

目的:通过基因克隆和体外转录,获得汉坦病毒汉滩型76118株及汉城型R22株S基因的RNA全长cRNA,为汉坦病毒病原学检测提供阳性定量标准品。方法:设计汉滩型76118株和汉城型R22株S基因克隆引物,PCR获得相应片段,分别克隆至含双启动子的PCRⅡ载体中,测序鉴定无误后,重组质粒分别经内切酶SpeⅠ、SacⅠ线性化,用T7 RNA聚合酶进行体外转录,产物经DNase处理、纯化后测定浓度,经RT-PCR验证。结果:获得汉滩型76118株及汉城型R22株S基因的cRNA片段,并可准确定量其拷贝数,76118株和R22株的质量浓度分别为80、17.58 ng/μL。结论:获得的cRNA样品可作为汉坦病毒核酸快速检测方法的阳性定量标准品。     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

A defective interfering RNA that contains a mosaic of a plant virus genome

A symptom-modulating RNA associated with tomato bushy stunt virus (TBSV) was investigated with respect to physical and biological properties. Linear RNA of approximately 396 nucleotides was packaged in viral coat protein and was dependent on TBSV for replication. Coinoculation of the small RNA with TBSV resulted in the attenuation of TBSV-induced symptoms and depression of virus synthesis in whole plants. Nucleotide sequence analysis revealed that the symptom-modulating RNA was derived from 5', 3', and internal segments of the TBSV genome. The identification of this symptom-modulating RNA as a co-linear deletion mutant of the helper virus genome establishes it as the first definitive defective interfering RNA (DI RNA) to be identified in association with a plant virus.     

A cytoplasmic polyhedrosis virus isolated from the pine processionary caterpillar, Thaumetopoea pityocampa

A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.     

Increased virulence of a mouse-adapted variant of influenza A/FM/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8.

To cause disease, influenza virus must possess several genetically determined abilities that mediate stages in pathogenesis. The virulent mouse-adapted variant A/FM/1/47-MA (FM-MA), derived from the avirulent A/FM/1/47 (FM) strain, had acquired mutations in genes that control virulence. The purpose of this study was to identify those genes that had mutated to result in increased virulence and to obtain viruses that differed in virulence because of differences in individual genome segments. The genes that had mutated to increase virulence were initially identified by genetic analysis of reassortants obtained by crossing FM-MA with the avirulent strain A/HK/1/68 (HK). FM-MA genome segments 4, 5, 7, and 8 were significantly associated with virulence, as determined by using the Wilcoxon ranked sum analysis. The role of FM-MA segments 4, 7, and 8 was confirmed by reintroduction of these genes into the parental strain, which also provided virus strains that differed in virulence because of mutations in individual genome segments. Segments 4, 7, and 8 were responsible for a 10(3.6)-fold increase in virulence that was proportioned 10(2.2)-, 10(0.7)-, and 10(0.8)-fold, respectively. The role of segment 5 could not be confirmed on transfer back into the parental strain because of reversion during preparation of such reassortants. The incidence of reversion was shown to be significantly associated with culturing of FM-MA in chicken embryo cells but was not associated with growth in MDCK cells. The genetic analysis of FM-MA suggests that adaptation to increased virulence is an incremental process that involves the acquisition of mutations in multiple genes, each of which plays an individual role in pathogenesis. The structural and functional properties of segments 4, 7, and 8 that control the virulence of FM-MA can now be determined by using viruses that differ in virulence because of mutations in these individual genome segments.     

Effects of the Number of Genome Segments on Primary and Systemic Infections with a Multipartite Plant RNA Virus

Multipartite plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 plants, the data support the IAH model, showing that the expression of virus genome segments by the host plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.