Map location of lactate dehydrogenase-elevating virus (LDV) capsid protein (Vp1) gene

Lactate dehydrogenase-elevating virus (LDV) is currently classified within the Togaviridae family. In an effort to obtain further information on the characteristics of this virus, we have begun to sequence the viral RNA genome and to map the virion structural protein genes. A sequence of 1064 nucleotides, which represents the 3' terminal end of the genome, was obtained from LDV cDNA clones. A 3' noncoding region of 80 nucleotides followed by two complete open reading frames (ORFs) were found within this sequence. The two ORFs were in different reading frames and overlapped each other by 11 nucleotides. One ORF encoded a protein of 170 amino acids and the other ORF, located adjacent to the 3' noncoding region of the viral genome, encoded a 114 amino acid protein. Thirty-three N-terminal residues were sequenced directly from purified LDV capsid protein, Vp1, and this amino acid sequence mapped to the ORF adjacent to the 3' noncoding region. The presence of overlapping ORFs and the 3' terminal map position of Vp1 indicate that LDV differs significantly from the prototype alpha togaviruses.     

Update and comparison of the immediate-early gene DNA sequences of two pseudorabies virus isolates

Differences between the immediate-early gene DNA sequences of two pseudorabies virus isolates (Indiana-Funkhauser and Ka) were resolved and confirmed. The deduced amino acid sequences showed that regions 2 and 4 have fewer changes than the rest of the molecules. These two conserved regions may be functionally important.     

Lactate dehydrogenase-elevating virus replication, maturation, and viral RNA synthesis in primary mouse macrophage cultures.

High titers of lactate dehydrogenase-elevating virus (LDV) are obtained after infection of 1-day primary peritoneal mouse macrophage cultures. The capacity of these cells to support LDV replication, however, progressively decreases as the cells age. Almost no replication occurs in 7-day cultures, in spite of the fact that the cells are viable and metabolically active, though nondividing. Mitotically active cultures of simian virus 40 (SV40)-transformed macrophages also fail to support LDV replication. The synthesis of LDV-specific RNA was studied by labeling infected 1-day macrophage cultures with [5-³H]uridine in the presence of actinomycin D. The synthesis of single-stranded 48 S viral RNA and RNase-resistant 27 S RNA commence between 4 and 5 hr after infection, and the first mature virions are released between 5 and 6 hr. The time period between synthesis of viral RNA and its appearance in mature extracellular virions is about 1.5 hr. The induction of viral RNA synthesis is completely inhibited by treatment of cells with cycloheximide 0.5 hr after infection.Electron microscopic examination of thin sections of infected macrophages shows that LDV matures by budding from the cytoplasm into intracytoplasmic vesicles. Mature virions seem to be rapidly released into the culture fluid by an as yet unknown mechanism.     

The extreme 5'-terminal sequences of sindbis virus 26 and 42 S RNA.

The messenger RNA species specified by Sindbis virus in infected cells, 26 and 42 S RNA, have been characterized with regard to their extreme 5′-terminal sequences. (Methyl-³H)-Labeled m⁷G in “caps” was used to detect cap-containing, and hence 5′-terminal, oligonucleotides after digestion with ribonuclease A, or ribonuclease T₁, plus phosphatase. Digests were fractionated by DEAE column chromatography, by cellulose acetate electrophoresis, and by DEAE paper electrophoresis. The latter two systems were also applied sequentially to ³²P-labeled samples to generate oligonucleotide fingerprints. The cap-containing oligonucleotide from T₁-plus-phosphatase digests of 26 S RNA was isolated and shown to be m⁷ GpppApUpG. The homologous oligonucleotide from such a digest of 42 S RNA behaved as if it were one pyrimidine residue larger, whereas cap-containing oligonucleotides from ribonuclease A-plus-phosphatase digests of the two RNA species had identical chromatographic and electrophoretic properties. We infer from these and earlier findings that Sindbis virus-specified 42 S RNA terminates in m⁷GpppApUpYpG. These results support the idea that 26 S RNA and 42 S RNA arise from two distinct and different initiation sites.     

The entire nucleotide sequences of two distinct TT virus (TTV) isolates (TJN01 and TJN02) remotely related to the original TTV isolates

Summary.  TT virus (TTV) has a wide range of sequence divergence by which it is classified into at least 16 genotypes. A TTV isolate of genotype 12 (TJN01) and another of genotype 13 (TJN02) were sequenced in the entire genome, and compared with the reported TTV isolates. TJN01 and TJN02 had genomic lengths of 3787 and 3794 nucleotides (nt), respectively, which were shorter by 66 and 59 nt than the prototype TTV isolate of genotype 1 (TA278). TJN01 and TJN02 shared the nucleotide sequence with TA278 merely in 53.9% and 55.2%, respectively. They possessed two major open reading frames (ORFs) and the noncoding region with a GC-rich region forming stem-loop structures, which are characteristic of TTV. However, their amino acid sequences in ORF1 were similar to that of TA278 in only 35.4 and 34.0%, respectively; TJN01 was 45.4% similar to TJN02. Comparison with TTV isolates of the same genotype identified hypervariable regions in ORF1 of TJN01 and TJN02, as in the prototype TTV of genotype 1. However, quasispecies were barely observed in them. Furthermore, sequences of hypervariable regions scarcely changed during 2–5.5 years in both TJN01 and TJN02. These results indicate that TTV of genotypes 12 and 13 are much different from the prototype TTV of genotype 1. Received January 24, 2000 Accepted March 22, 2000     

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations

Summary Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV₃) and chilo iridescent virus (CIV), two other well known viruses of the familyIridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A₂. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.     

Unique RNA 2 sequences of two Brazilian isolates of Pepper ringspot virus,a tobravirus

Pepper ringspot virus (PepRSV) is a tobravirus reported only in Brazil. Here, the sequences of the complete RNA 2 segments and the 3′ end of the RNA 1 genomic regions of two new isolates from tomato plants were analyzed. The main ORF encodes the CP gene as other tobraviruses and termed ORF 1 of RNA 2. The second ORF was found only in one of the new isolates, although this gene was absent in the type isolate, CAM (collected in the 1960’s). Interestingly, this ORF 2 gene did not show any nucleotide and amino acid sequence similarities with known 2b genes of tobraviruses, an essential gene of tobraviruses for nematodes-transmission. The 5′UTR sequence of RNA 2 segment of CAM isolate was previously reported showing two impaired direct repeats; however, the direct-repeats were absent in these new isolates. An additional ORF was predicted upstream of the CP gene. This putative protein possessed a transmembrane domain similar to the ORFN1 of RNA 2 of Tobacco rattle virus SYM isolate, although there was no sequence similarity. This is the first report on the diversity of the RNA 2 sequences of PepRSV.     

Complete genomes of two Human hepatitis A virus isolates from China: analysis and comparison with other isolates

Complete sequences of the genomes of two wild type (wt) Human hepatitis A virus (HHAV) isolates, LU38 and LY6 from China were determined and compared with those of wt HHAV isolates AH1, AH2, AH3, FH1, FH2, FH3, GBM, HM175, LA and MBB. The genomes of both LU38 and LY6 consisting of 7477 nucleotides (nts) contained a 5'-non-translated region (5'-NTR, 733 nts), an open reading frame (ORF, 6681 nts), and a 3'-NTR (63 nts) followed by a poly(A)-tail. It encoded a polyprotein of 2227 amino acids (aa) Sequence comparison showed that LU38 shared the highest identities of 98.1% for nt (140 differences) and 99.2% for as (17 differences) with AH1, and the lowest identities of 91.4% for nt (741 differences) with HM175 and 98.1% for aa (43 differences) with GBM. LY6 shared the highest identities of 97.4% for nt (196 differences) and 98.7% for aa (28 differences) with H1 and the lowest identities of 91.2% for nt (642 differences) with HM175 and 97.7% for as (51 differences) with GBM. The subgenotyping revealed that the LU38 and LY6 isolates are of IA subgenotype. The phylogenetic analysis showed that LU38 is closest to AH1 and the LY6 to FH3, suggesting that the epidemiological link of hepatitis A (HA) had developed in China and Japan.     

The RNAs of cucumoviruses: 3'-terminal sequence analysis of two strains of tomato aspermy virus

The sequences of 189 residues from the 3' terminus of three RNAs of one strain and two RNAs of another strain of tomato aspermy virus have been determined; there was almost complete sequence homology between the RNAs. A base-paired transfer RNA-like structure is proposed for tomato aspermy virus RNAs which is similar in many aspects to the structure proposed for the 3'-terminal 172 residues of RNA 4 of the Q-strain of cucumber mosaic virus (R. H. Symons, Nucleic Acids Res.7, 825-837, 1979). These 172 residues of cucumber mosaic virus RNA 4 can be aligned to show 73% sequence homology with the 3'-terminal 189 residues of the tomato aspermy virus RNAs.     

Complete nucleotide sequences of two isolates of cherry green ring mottle virus from peach (Prunus persica) in China

Two complete nucleotide sequences of cherry green ring mottle virus (CGRMV) isolated from peach in Hebei (Hs10) and Fujian (F9) Provinces, China, were determined. Five open reading frames (ORFs) were found in the genomes of both isolates. The F9 and Hs10 isolates shared 82.2 % and 83.4-94.4 % nucleotide sequence identity, respectively, with two CGRMV isolates from cherry. Analysis of the nucleotide and amino acid sequences from the five ORFs of both isolates showed that Hs10 shares the greatest sequence identity with P1A (GenBank AJ291761) from cherry. Phylogenetic analysis indicated that CGRMV isolates from peach and cherry are closely related to members of the genus Foveavirus.     

Immunization with inactivated lactate dehydrogenase-elevating virus followed by virus infection favors the selection of hybridomas synthesizing antibodies cross-reacting with intermediate filaments and the LDV envelope protein VP3

Autoantibodies against Golgi antigen and a tumor surface antigen (TSA) of aetiologically unrelated murine cell transformants that develop in immunocompetent mice as early as 6-7 days after treatment with live LDV do not appear after immunization with inactivated virus. However, combination of immunization with inactivated LDV and treatment with live LDV enhances the production of autoantibodies against determinants of intermediate filaments. The basis of the stimulation of this group of autoantibodies is at least in part due to antigenic mimicry between the envelope protein VP3 of LDV and determinants of intermediate filaments, since a panel of monoclonal antibodies cross-reacts with both.     

Nucleotide sequences of Australian isolates of the feline immunodeficiency virus: comparison with other feline lentiviruses

Summary Proviral DNA from four Australian isolates of feline immunodeficiency virus (FIV) was amplified by PCR and the nucleotide sequence determined for two conserved regions withingag (p 15/p 24) andpol (RT) genes. Comparison with the nucleotide and deduced amino acid sequence of two previously described U.S. isolates from California (Petaluma and PPR), and a third from Maryland (MD) as well as the Japanese isolate TM 2, revealed a close similarity between the Australian and Californian isolates with 95–97% nucleotide and 96–99% amino acid homologies. By contrast, the Maryland and Japanese isolates were more distantly related with only 84–87% nucleotide and 90–94% amino acid homology with either the Australian or Californian isolates. The relationship of the Australian FIV isolates to other domestic isolates as well as eight lentiviral isolates from wild felidae (panthers) published previously, was investigated further by constructing a phylogenetic tree based on thepol sequence. This revealed two subgroups of FIV, an Australian/Californian group and a less tightly clustered Maryland/Japanese group. These results suggest that the genomic variability of FIV is reflected by more than simply geographic distance. Furthermore, the relative genetic homogeneity found between Australian isolates suggest a shorter period of evolution of the virus in Australia than in North America.     

Studies on the relationship between 5' leader sequences and initiation of translation of adenovirus 2 and simian virus 40 late mRNAs

The question of whether or not 5′ leader sequences might confer characteristic translational properties to the mRNAs to which they are spliced was studied using two complementary experimental situations with respect to the composition of mRNA molecules. Late in adenovirus type 2 (Ad-2) infection of human cells, a common tripartite 5′ leader sequence is spliced to mRNA sequences coding for at least 13 different late viral proteins. This results in different mRNAs with identical 5′ leader sequences. In the second experimental system, identical coding sequences are spliced to different 5′ leader sequences with different caps. Wild-type SV40 VP1 mRNA (16 S) contains a 5′ leader sequence coded for at position 72–76 on the SV40 genome; in the SV40 mutant dl-808 part of the sequences in this region, including the sequence coding for cap, is deleted. The relative ability of these mRNAs to direct incorporation of radioactive amino acids into their respective polypeptide products was assayed in vivo under conditions of hypertonic initiation block (HIB) or in the presence of low concentrations of cycloheximide. Messenger RNAs with identical 5′ leader sequences were observed to exhibit very different responses in both experimental situations. Conversely, similar responses to HIB were observed for VP1 mRNAs with different 5′ leader sequences. It thus appears that the presence of specific sequences per se between the 5′ cap and the initiation codon does not alone confer characteristic initiation properties as measured indirectly by perturbation of the in vivo environment.     

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.     

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Complete nucleotide sequences of the genomes of two isolates of apple chlorotic leaf spot virus from peach (Prunus persica) in China

The complete nucleotide sequences of two isolates of apple chlorotic leaf spot virus (Z1 and Z3) collected from peach in Henan Province, China, were determined. The genomes of both Z1 and Z3 were found to contain three open reading frames (ORFs). Sequence analysis showed that genomic sequences of Z1 and Z3 isolates shared 67.4%-82.9% and 67.2%-82.6% identity, respectively, with the other eight isolates of ACLSV that have been reported previously. Based on the putative amino acid sequences of the products of the three ORFs, Z1 and Z3 isolates showed the greatest identity to isolate PBM1 (GenBank accession number AJ243438) from plum and the least identity with isolate Ta Tao5 (GenBank Accession Number: EU223295) from peach. Considering the low level of sequence identity between Z1/Z3 isolate and Ta Tao5 isolate, two types of ACLSV may exist in peach.     

Comparative transmission of two cucumber mosaic virus isolates by two color morphs of Acyrthosiphon pisum (Harris)

Cucumber mosaic virus (CMV) is one of the most important legume-infecting viruses, which is transmitted effectively by pea aphid Acyrthosiphon pisum (Harris) (Hem: Aphididae). Transmission efficiency of two CMV isolates (As and Kh from cowpea and bean hosts, resp.) by red and green color morphs of pea aphid were evaluated on bean plants. Triple-antibody sandwich ELISA (TAS-ELISA) using CMV-specific monoclonal antibodies revealed that both CMV isolates belonged to the serotype II. Bean plants inoculated by viruliferous aphids were assayed by double-antibody sandwich ELISA (DAS-ELISA) at 16 days post inoculation (dpi). The results showed that the transmission rate of CMV-As by the red morph was significantly higher than by the green morph, resulting in significantly higher transmission rate of the virus (As + Kh) by the red morph than by the green morph, with p≤ 0.1. Similarly, the efficiency of CMV transmission by the red morph of A. pisum was higher than the efficiency of transmission by the green morph. The higher transmission rate and efficiency of CMV by red pea aphid would be important in the epidemiology. Based on these results, we hypothesize that the transmission efficiency of CMV is affected more by the difference in transmission determinants of A. pisum color morphs than by the sequence of virus coat protein determinants. Keywords: Aphididae; Bromoviridae; color polymorphism; transmission efficiency.