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C S Schmaljohn

Publications and source records attributed to C S Schmaljohn.

At least 37 records · Page 2Linked to original sources
PubMed1995

Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome.

We report the complete nucleotide sequence of the M and the S genome segments and a portion of the L segments of two hantavirus isolates from Peromyscus maniculatus trapped in eastern California. The isolates, Convict Creek 107 and 74 (CC107 and CC74) are genetically similar to viruses known to cause hantavirus pulmonary syndrome in New Mexico. CC107 and CC74 each have an M segment consisting of 3696 nucleotides with a coding potential of 1140 amino acids in the virus complementary-sense RNA (cRNA). The S segments of CC107 and CC74 are 2083 and 2047 nucleotides long, respectively, and each has an ORF in the cRNA capable of encoding a protein of 428 amino acids. Unusually long 3' noncoding regions of 757 and 721 nucleotides follow the S segment ORF of CC107 and CC74, respectively, and include numerous imperfect repetitive sequences. Whereas the M and S segments of any given hantavirus typically appear to diverge at comparable rates from homologous genes of any other hantavirus, CC107 and CC74 have M segments that differ by only 1% from one another but S segments that differ by 13%. After trivial explanations are rendered improbable, i.e., by consideration of the genetics of closely and distantly related hantaviruses, the most likely explanation for our data is that hantavirus genome segment reassortment occurred within rodent populations in California.

Animals↗
PubMed1995

A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus.

To investigate the ability of a vaccinia virus-vectored vaccine expressing the M and the S segments of Hantaan (HTN) virus (C. S. Schmaljohn, S. E. Hasty, and J. M. Dalrymple, Vaccine 10:10-13, 1992) to elicit a protective immune response against other hantaviruses, we vaccinated hamsters with the recombinant vaccine and challenged them with HTN, Seoul (SEO), or Puumala (PUU) virus. Neutralizing antibodies to HTN virus were found in all vaccinated hamsters both before and after challenge. Neutralizing antibody titers to SEO virus were present at low levels or were undetectable after two immunizations with the vaccine but were positive in all vaccinated hamsters after challenge with SEO virus and were also positive in control animals that were not challenged. Neutralizing antibodies to PUU virus were observed only in hamsters previously challenged with PUU virus. To assay for virus in the blood and tissues of the hamsters, we developed a nested reverse transcriptase (RT)-PCR with cross-reactive outer primers and serotype-specific inner primers. The RT-PCR specifically detected as little as 1 PFU of virus in serum containing high-titer neutralizing antibodies and was more sensitive than immunofluorescent antibody staining for detecting virus in lung and kidney specimens of infected hamsters. By using the RT-PCR, we found that vaccinated hamsters, challenged with HTN or SEO virus, neither were viremic nor had evidence of virus in their lungs or kidneys. In contrast, vaccinated hamsters challenged with PUU virus were viremic and had PUU virus-specific nucleic acid in their organs.

Animals↗
PubMed1995

Short report: prevalence of hantavirus infection in rodents associated with two fatal human infections in California.

Rodents living near two fatal human cases of hantavirus pulmonary syndrome in California were surveyed for evidence of hantavirus infection. Seventeen (15%) (14 Peromyscus maniculatus and one each of P. truei, Eutamias minimus, and Microtus californicus) of 114 rodents tested had evidence (enzyme-linked immunosorbent assay or polymerase chain reaction) of hantavirus infection. This suggests that Peromyscus mice, and P. maniculatus in particular, may be the reservoir for the virus causing this newly recognized disease in California, as previously reported for New Mexico and Arizona.

Adult↗
PubMed1994

Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae.

We examined the serological relationships among 32 hantavirus isolates collected from numerous geographic regions and hosts. We prepared rat immune sera to each virus and used these sera in hemagglutination inhibition (HAI) tests, enzyme-linked immunosorbent assays (ELISA), and plaque-reduction neutralization tests to establish the antigenic relationships among the isolates. Our studies included viruses that had been partially characterized previously, as well as isolates for which little or no serological data were available. Our results indicate that, in addition to the four established serological groups of hantaviruses, represented by Hantaan, Seoul, Puumala, and Prospect Hill viruses, there exist at least two other distinct groups of hantaviruses. These groups are represented by Thailand 749 virus, an isolate from Bandicota in Thailand, and by Thottapalayam virus, an isolate from Suncus in India. To compare more closely the antigenic relationships of the isolates to prototype Hantaan virus, we examined the reactivities of Hantaan G1- or G2-specific monoclonal antibodies with the envelope proteins of a number of the isolates. We found that several epitopes defined by monoclonal antibodies to the G2 protein were highly conserved as detected by HAI tests and ELISA. Almost all of the isolates could be neutralized by at least one G2-specific monoclonal antibody. In contrast, epitopes defined by G1 monoclonal antibodies were conserved only among Hantaan-like viruses.

Animals↗
PubMed1994

Phylogenetic analyses of virus isolates in the genus Hantavirus, family Bunyaviridae.

We constructed a phylogenic tree by pairwise comparison of the deduced amino acid sequences encoded in the medium (M) genome segments of 13 hantaviruses. Five distinct branches were identified, four of which are represented by Hantaan, Seoul, Puumala, and Prospect Hill viruses. These groups corresponded to those previously established by serological and genetic means. In addition, we present sequence information for the M segment of Thailand virus and demonstrate that it represents a unique, fifth branch on the dendrogram. We show that a tree nearly identical to that constructed from the deduced amino acids encoded in the M segments of these viruses can be constructed by comparing nucleotide sequences of a 333-bp region of the M segments of the same hantaviruses. This region can be amplified, for most hantaviruses, by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques, using a single primer pair. By using this method, we constructed a consensus tree for 30 hantaviruses, including 15 isolates for which we PCR-amplified and sequenced the 333-bp region. In addition to the five branches described above, we identified a sixth unique hantavirus group represented by Dobrava virus, an Apodemus isolate from Slovenia. Although we were unable to PCR-amplify the M segment of Thottapalayam virus, a distantly related hantavirus isolated from a shrew captured in India, we were able to amplify, clone, and sequence a portion of the small (S) segment of that virus. When we compared our Thottapalayam sequence to corresponding S segment sequences of other hantaviruses, the results indicated that this virus may represent a seventh, distinct group of hantaviruses.

Animals↗
PubMed1994

Antigenic and molecular characterization of hantavirus isolates from China.

Hemorrhagic fever with renal syndrome (HFRS) is caused by certain viruses in the genus Hantavirus, family Bunyaviridae, and is a major public health problem in China. By using molecular and serological tests, we characterized 15 hantaviruses isolated either from patients with HFRS or from rodents captured in endemic areas of China. By cross plaque-reduction neutralization tests performed with rabbit immune sera, we identified two serologically distinct groups of viruses, comprised of those related to Hantaan virus, and those related to Seoul virus. To study the genetic relationships among these viruses, we amplified a 330 base pair region of the medium (M) genome segment of each isolate by reverse transcription and polymerase chain reaction (PCR) and compared the nucleotide sequences to those of other, well-characterized hantaviruses. In addition, we PCR-amplified and analyzed the entire coding region of the small (S) genome segment of each isolate by restriction enzyme digestion with a battery of enzymes. The results of our genetic analyses of both the M and S segments of these isolates confirmed our serological data, indicating that Hantaan and Seoul viruses co-circulate in endemic disease regions of China. We constructed a phylogenetic tree based on multiple alignment of the partial M segment sequences. The resulting dendrogram distinguished three genetic subtypes of Hantaan viruses and one type of Seoul virus.

Animals↗
PubMed1994

Expression strategy of the M genome segment of Hantaan virus.

The medium (M) genome segment of Hantaan virus encodes the envelope glycoproteins, G1 and G2, in a continuous open reading frame with a gene order of 5'-G1-G2-3' with respect to the virus-complementary sense RNA. Because potential translation initiation codons and amino acids constituting typical signal sequences precede both the G1 and G2 genes, we sought to determine if G1 and G2 can be expressed independently. To investigate translational requirements for G1 and G2, we constructed M segment genes in which portions of the coding information were mutated or deleted, and transiently expressed these genes in eukaryotic cells by using a vaccinia virus/T7 RNA polymerase system. We found that G2 expression can occur by ribosomal access to the translation initiation codon preceding the G2 signal sequence (nucleotides 1934-1936), but that other upstream AUG codons cannot be used as efficiently. The presence of this codon, however, was not required for G2 expression because changing nucleotides 1934-1936 to CUG, GCG or AUG did not abrogate expression of G2. We also found that leaky ribosomal scanning, rather than internal initiation of translation was the most likely explanation for the observed independent translational initiation of G2, but that not all upstream, in-frame AUGs could serve as initiator codons. To assess the requirement for a continuous open reading frame for G1 and G2 expression, we expressed a gene which had G1 and G2 coding information in different reading frames. Although G1 was expressed at apparently normal levels, little or no G2 was expressed. In contrast, only G2 was expressed from a gene in which the carboxy-terminal G1 coding information was deleted and the remaining, truncated G1 was placed out of frame with respect to G2. These data suggest that reinitiation of translation may occur under some, but not all, circumstances when the polyprotein coding information is perturbed. Our results are consistent with biogenesis of G1 and G2 primarily or entirely according to the ribosomal scanning model.

Animals↗
PubMed1993

Epitope mapping studies with neutralizing and non-neutralizing monoclonal antibodies to the G1 and G2 envelope glycoproteins of Hantaan virus.

Epitopes recognized by three G1-specific and two G2-specific neutralizing monoclonal antibodies to Hantaan virus were mapped by sequence analyses of the complete M genome segments of neutralization escape variant viruses. For each variant, we detected nucleotide sequence substitutions which resulted in a single amino acid change in either the G1 or G2 protein. Serological properties of the variant viruses correlated with changes identified by nucleotide sequence analyses. To map epitopes recognized by three G1-specific and six G2-specific, non-neutralizing monoclonal antibodies, we prepared genes, truncated at the carboxy terminal coding regions of G1 or G2, and expressed them with baculovirus recombinants or transiently in a vaccinia/T7 RNA polymerase system. Reactivities of the monoclonal antibodies with the truncated proteins were monitored by immune precipitation of the radiolabeled, truncated glycoproteins. We determined that all three of the G1-specific antibodies reacted with truncated proteins, which retained the amino terminal one-third of G1, but lost reactivity with shorter G1 proteins. The G2-specific antibodies only recognized G2 proteins, which retained approximately 80% of the G2 gene.

Amino Acid Sequence↗
PubMed1993

Nucleotide and deduced amino acid sequences of the M and S genome segments of two Puumala virus isolates from Russia.

Hemorrhagic fever with renal syndrome (HFRS) is caused by viruses in the Hantavirus genus, family Bunyaviridae. Three serologically distinct hantaviruses, Hantaan, Seoul and Puumala viruses, are known to cause HFRS. We report here, for the first time, gene sequences of two human Puumala virus isolates, P360 and K27, obtained in an HFRS endemic region of the former Soviet Union. We compared the nucleotide sequences and the derived amino acid sequences of their gene products to a Puumala virus isolate from rodents.

Amino Acid Sequence↗
PubMed1993

Expression of non-conserved regions of the S genome segments of three hantaviruses: evaluation of the expressed polypeptides for diagnosis of haemorrhagic fever with renal syndrome.

Haemorrhagic fever with renal syndrome (HFRS) is a serious and often fatal disease caused by viruses in the Hantavirus genus of the family Bunyaviridae. We expressed the entire coding region of the small (S) genome segments of three serologically distinct hantaviruses as soluble proteins in Escherichia coli and evaluated the expressed nucleocapsid proteins (NPs) as antigens for diagnosis of HFRS. We also prepared novel diagnostic antigens by expressing truncated genes from which we deleted amino acid coding regions that were highly conserved among the three viruses. These antigens were analysed for their potential to detect and differentiate between antisera to various hantaviruses by ELISA. ELISA results obtained with HFRS patient sera or with sera from naturally or experimentally infected animals indicate that homologous antigens and antisera reacted to high titre. The truncated NPs were more specific than the complete NPs in distinguishing between possible aetiological agents of HFRS. Our findings demonstrate that prokaryotic expression of portions of the NPs of specific hantaviruses can be used to generate, readily and efficiently, large quantities of antigen that is both sensitive and specific in diagnostic assays for HFRS.

Animals↗
PubMed1993

Molecular and antigenic characterization of HV114, a hantavirus isolated from a patient with haemorrhagic fever with renal syndrome in China.

The relationship of a Hantaan-like virus (HV114), isolated from a patient with haemorrhagic fever with renal syndrome in Hubei Province, People's Republic of China, to other pathogenic hantaviruses was evaluated by cross-neutralization studies and nucleotide sequence analysis of the M genome segment. Plaque reduction neutralization assays indicated that HV114 is closely related to prototype Hantaan (HTN) virus, strain 76-118, which was originally isolated from an Apodemus field mouse in Korea. Comparison of the M genome segments of HTN 76-118 and HV114 revealed sequence identity of 84.7% and 95.4% for nucleotides and deduced amino acids, respectively. These data demonstrate that HV114 and 76-118 are two closely related but different isolates of HTN virus, establishing the scientific basis for testing and future use in China of a recombinant vaccine expressing the genome of HTN virus strain 76-118.

Amino Acid Sequence↗
PubMed1992

Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex.

To study the intracellular transport and targeting to the Golgi complex of the membrane glycoproteins G1 and G2 of Hantaan virus, we have expressed them together and separately using recombinant vaccinia viruses. When expressed from the same recombinant vaccinia virus, G1 and G2 were localized to the Golgi complex as analyzed by both immunofluorescence and subcellular fractionation. However, when the glycoproteins were expressed from separate recombinant viruses, both proteins remained in the endoplasmic reticulum. Using several monoclonal antibodies, it was found that G1 expressed alone did not acquire its correct conformation. Finally, if cells were coinfected with G1- and G2-expressing recombinant viruses, the proteins were again targeted to the Golgi complex. The N-linked glycans remained in all cases largely endoglycosidase-H sensitive. With none of the recombinant viruses were expression of the glycoproteins observed on the cell surface. Neither did chasing in the presence of cycloheximide result in the surface expression of G1 or G2. Our results indicate that for transport out of the endoplasmic reticulum and proper targeting to the Golgi complex, the two glycoproteins have to be coexpressed. The most likely interpretation is that G1 and G2 have to interact with each other in the endoplasmic reticulum in order to become transport competent.

Cell Compartmentation↗
PubMed1992

Cloning and sequence analysis of the genes encoding the nonstructural proteins of Langat virus and comparative analysis with other flaviviruses.

Langat virus, a member of the family Flaviviridae is antigenically very similar to highly pathogenic tick-borne encephalitis viruses. We cloned and sequenced the complete nonstructural gene-coding region of Langat virus (strain TP21) and compared the deduced amino acid sequences of each nonstructural protein to those of other flaviviruses. By alignment with the reported amino acid sequences of the nonstructural proteins of several flaviviruses, we were able to predict proteolytic cleavage sites and identify sequence motifs, which are highly conserved among flaviviruses. Sequence similarity calculations revealed that the NS3 and NS5 proteins are the most highly conserved of the flavivirus nonstructural proteins. The NS3 and NS5 proteins of Langat virus contained specific peptide sequences that have been demonstrated to be associated with helicase or polymerase activities, respectively. The NS1 protein of Langat virus displayed complete homology of potential N-linked glycosylation sites and cysteine residues with the NS1 proteins of other tick-borne flaviviruses, suggesting a highly conserved NS1 protein structure. The data presented in this report serve to complete the entire sequence of the Langat virus-coding region and provide the basis for comparison of this naturally attenuated virus to the other highly virulent tick-borne flaviviruses.

Amino Acid Sequence↗
PubMed1992

Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome.

Two vaccinia-vectored candidate vaccines for haemorrhagic fever with renal syndrome were prepared by inserting cDNA, representing the medium (M) genome segment, or the M and the small (S) genome segments of Hantaan virus into the thymidine kinase gene of the Connaught vaccine strain of vaccinia virus. In the single recombinant, the M segment was placed under control of the vaccinia virus 7.5 kDa promoter. In the double recombinant, the M and S segments were placed under control of the vaccinia virus 7.5 kDa and 11 kDa promoters, respectively. An immunoplaque assay technique was developed to select recombinants without the need for expression of irrelevant genes or use of potential mutagens. Proteins indistinguishable from authentic viral envelope glycoproteins and nucleocapsid protein were observed by immunoprecipitation with antibodies to Hantaan virus. The recombinant expressing both the M and the S segments was selected for further development and testing as a human vaccine.

DNA, Viral↗
PubMed1990

Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome.

Three serologically distinct groups of hantaviruses have been associated with severe, moderate, and mild forms of hemorrhagic fever with renal syndrome (HFRS). To gain a better understanding of the genetic variation among these viruses, we cloned and sequenced the M and the S genome segments of Sapporo rat virus, an etiologic agent of moderate HFRS, and compared the predicted gene products to those of Hantaan virus, and the Hällnäs strain of Puumala virus, which are etiologic agents of severe and mild HFRS, respectively. The SR-11 S segment consisted of 1769 nucleotides and had an open reading frame (ORF) in the virus-complementary sense RNA with a coding capacity of 429 amino acids. Deduced amino acids from the SR-11 S segment ORF displayed 83% homology with those of Hantaan nucleocapsid (N) protein. Comparison of the S segment ORFs of all three viruses revealed 58% homology. No evidence for additional nonstructural protein(s) encoded by the SR-11 S segment was obtained. The SR-11 M segment consisted of 3651 nucleotides and had an ORF in the virus-complementary sense RNA with a coding capacity of 1134 amino acids. Amino acid sequences predicted from the SR-11 M segment ORF were 75% homologous with those encoding Hantaan G1 and G2 envelope glycoproteins. Comparison of the deduced amino acid sequences of the M segment ORFs of SR-11, Hantaan, and Hällnäs viruses revealed a 43% homology for amino acids constituting the G1 proteins and a 55% homology for amino acids constituting the G2 proteins of the three viruses. The envelope proteins of SR-11 virus were localized within the M segment ORF by amino-terminal sequence analysis of purified G1 and G2. G1 initiated at amino acid 17 and G2 at amino acid 647 within the ORF. Five potential asparagine-linked glycosylation sites were identified in the SR-11 G1 coding sequences, four of which were conserved between Hantaan and SR-11 viruses and three of which were conserved among all three viruses. One potential glycosylation site was identified in the SR-11 G2 coding sequences and was conserved among Hantaan, SR-11 and Hällnäs viruses. Cysteine residues were highly conserved within the M segment ORFs of all three viruses, suggesting a similar structure and function of the G1 and G2 proteins.

Amino Acid Sequence↗
PubMed1990

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

Animals↗