Frederick J. Fuller

Characterization of infectious molecular clones of equine infectious anaemia virus

We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences. We further investigated LTR sequence variation in the PV stock using PCR amplification to obtain additional LTR clones from infected FEK cells and from peripheral blood mononuclear cells (PBMCs) from animals experimentally infected with PV. Sequence analysis of resulting clones indicates a selection for different LTR populations in pony PBMCs compared to FEK cells. Finally, we observed that the cloned EIAV proviruses did not remain infectious when maintained in a derivative of pBR322. However, two proviruses have been stably maintained in a low copy number vector (pLG338-SPORT).

Bunyavirus Nucleoprotein, N, and a Non-structural Protein, NSS, Are Coded by Overlapping Reading Frames in the S RNA

It has been shown previously, by sequence analysis of the S RNA segment of snowshoe hare (SSH) bunyavirus, that two overlapping open reading frames in the viral complementary sequence code for proteins with molecular weights of 26.8 X 10(3) and 10.5 X 10(3) respectively. In addition to the viral nucleocapsid (N) protein, which is coded by the S RNA, analyses of parental and reassortant bunyavirus-infected cell extracts have shown that the viral S RNA and M RNA species each code for non-structural proteins (NSS and NSM, respectively). In the present report, in vitro translation analyses of the S mRNA species recovered from virus-infected cells indicate that a single size class of mRNA directs the synthesis of N and NSS. Compositional analyses of selected tryptic peptides of N and NSS have provided proof that N is the product of the first open reading frame, and NSS the product of the second.

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10.

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus.

Abstract The 3′ end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3′ untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3′ UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3′ UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3′ UTRs provide additional evidence that these avian coronaviruses are closely related.

Interference between bunyaviruses in Aedes triseriatus mosquitoes

Inhibition of the replication of alternate California serogroup bunyaviruses in Aedes triseriatus mosquitoes has been observed for mosquitoes previously infected with La Crosse (LAC) virus. By contrast, prior infection of mosquitoes with LAC virus did not interfere significantly with the subsequent infection and replication of Guaroa bunyavirus (Bunyamwera serogroup), or heterologous viruses such as West Nile flavivirus, or vesicular stomatitis rhabdovirus.

Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strains neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.

Sequence Analysis of the Matrix/Nucleocapsid Gene Region of Turkey Coronavirus

A reverse transcriptase, polymerase chain reaction (RT-PCR) procedure was used to amplify a segment of the genome of turkey coronavirus (TCV) spanning portions of the matrix and nucleocapsid (MN) protein genes (approximately 1.1 kb). The MN gene region of three epidemiologically distinct TCV strains (Minnesota, NC95, Indiana) was amplified, cloned into pUC19, and sequenced. TCV MN gene sequences were compared with published sequences of other avian and mammalian coronaviruses. A high degree of similarity (>90%) was observed between the nucleotide, matrix protein, and nucleocapsid protein sequences of TCV strains and published sequences of infectious bronchitis virus (IBV). The matrix and nucleocapsid protein sequences of TCV had limited homology (<30%) with MN sequences of mammalian coronaviruses. These results demonstrate a close genetic relationship between the avian coronaviruses, IBV and TCV.

Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

Complete nucleotide sequence of the tick-borne, orthomyxo-like Dhori/Indian/1313/61 virus nucleoprotein gene

The complete nucleotide sequence of the fifth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Ind/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. The fifth RNA contains 1479 nucleotides and can code for a protein of 477 amino acids with a molecular weight of 53,679 Da. The RNA 5 protein of the Dhori/Ind/1313/61 virus possesses five short regions (16-26 amino acids) which share a high degree (50-59%) of amino acid sequence homology with a computer-aligned consensus sequence of the influenza nucleoprotein gene family. These and other structural features of the RNA 5 protein suggest that RNA 5 of Dhori viruses codes for the nucleoprotein. The data also suggest that Dhori viruses are orthomyxoviruses, but that they are more distantly related to the influenza viruses than type A, B, and C viruses are to each other.

Synthesis and processing of sindbis virus nonstructural proteins in vitro

Abstract Polyadenylated 42 S genome RNA of Sindbis virus was extracted from purified virions, selected by binding to oligo(dT)-cellulose, and used to direct cell-free protein synthesis in nuclease-treated reticulocyte lysates. The translation products comprised a series of polypeptides which included distinct species with apparent molecular weights of 250, 205, 155,145, 89, 76, and 60 × 10 3 . Their interrelationships were examined by analysis of their kinetics of synthesis, peptide maps, and incorporation of radioisotopic label from formyl[ 35 S]methionyl-tRNA f . Polypeptides p89, p76, and p60 were unique, nonoverlapping, and stable. However, their sequences were contained in one or more of the larger products p250, p205, p145, which comprised a set of overlapping polypeptides that shared a common N-terminus. Radioisotope chase experiments showed that p250, p205, p155, and p145 were unstable; that p89, p76, and p60 were generated by proteolytic processing of larger precursors; and that the RNA regions encoding these three stable products were translated in the order: p60, p89, p76. On the basis of their electrophoretic mobilities and peptide maps, p89 and p76 were indistinguishable from two polypeptides found in Sindbis virus-infected cells and are candidates to be components of the Sindbis virus RNA-dependent RNA polymerases. When translation was performed under conditions that were designed to inhibit proteolysis, the accumulation of p89 and p76 was prevented, and that of p60 was severely inhibited. Under certain translation conditions, heterodisperse products larger than p250 accumulated. Even after thermal denaturation of RNA, only small amounts of the viral structural proteins were synthesized, indicating that the 3′-terminal third of the 42 S RNA genome was essentially unavailable for independent translation.