A murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells

Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of these viruses from the same cells, we constructed a porcine LLC-PK1 cell line stably expressing the recombinant MHV receptor cDNA (LMR cells). The MHV and TGEV receptor glycoproteins were shown by immunofluorescence to appear at the surface of the cells and to be functional so that the cells were susceptible to both MHV and TGEV infection. Both coronaviruses entered polarized LMR cells only through the apical surface. Remarkably, while the cells remained susceptible to TGEV for long periods, infectability by MHV decreased with time after plating of the cells onto filters. This was not due to a lack of expression of the MHV receptor, since this glycoprotein was still abundant on the apical surface of these cells. TGEV and MHV appeared to exit LMR cells from opposite sides. Whereas TGEV was released preferentially at the apical membrane, MHV was released preferentially at the basolateral surface. These results show that vesicles containing the two coronaviruses are targeted differently in LMR cells. We propose that the viruses are sorted at the Golgi complex into different transport vesicles that carry information directing them to one of the two surface domains. The apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in virus spread found between TGEV and MHV in their respective natural hosts, the former causing mainly a localized enteric infection, the latter spreading through the body to other organs.     

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds

Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2-99.5% to the vaccine virus RespPRRS and 99.0-99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus.     

Isolation of porcine respiratory coronavirus from pigs affected with porcine reproductive and respiratory syndrome

Four cytopathogenic viruses were isolated in CPK cells derived from porcine kidneys from tonsils and lungs of 3 of 15 pigs affected with porcine reproductive and respiratory syndrome virus. Physicochemically and morphologically, the isolates were similar to a coronavirus. The isolates were not distinguished from transmissible gastroenteritis virus (TGEV) by a neutralization test using polyclonal antibodies, but differentiated from TGEV by monoclonal antibodies capable of discriminating between TGEV and porcine respiratory coronavirus (PRCV), indicating that the isolates were PRCV. In a serological survey of 30 serum samples each collected from about 50 days old pigs in the 2 affected farms, 29 (97%) and 15 (50%) sera were positive for neutralizing antibody against the isolate with the titers ranging from 2 to 64, respectively.     

Renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents

cDNAs of potyviruses from Allium plants cultivated in different parts of the world were cloned by RT-PCR with a common primer for amplifying the 3' terminal genomic RNAs of onion yellow dwarf virus (OYDV), leek yellow stripe virus (LYSV) and, probably, of closely related potyviruses. Their nucleotide sequences bearing the viral coat protein (CP) gene and the 3' non-coding sequence were determined and compared. The degree of their sequence similarities clearly differentiated the respective viruses into 3 groups, namely OYDV "garlic-type", "wakegi-type" and LYSV group. The "garlic-type" included all the garlic isolates and two Indonesian shallot isolates. The "wakegi-type" group consisted of the isolates from Indonesian shallot and previously reported ones from Japanese Allium plants excluding garlic. The LYSV group was represented by LYSV isolates from garlic and leek. The CP sequences of LYSV group viruses differed from those of OYDV "garlic-type" and "wakegi-type" viruses (less than 60% similarities). In contrast, the sequence similarities between the OYDV "wakegi-type" and "garlic-type" isolates were 73.5 to 76.7%, suggesting they were closely related but should be discriminated as distinct species. These findings indicate that at least three distinct potyviruses, clearly distinguishable by the viral CP sequence, are present in Allium species. Finally, we concluded that the "garlic-type" viruses correspond to the typical OYDV and the "wakegi-type" viruses represent the viruses previously identified as Welsh onion yellow stripe virus (WoYSV) and shallot yellow stripe virus (SYSV). We propose the name wakegi yellow dwarf virus (WYDV) for the "wakegi-type" viruses.     

Social phobia subtypes in the National Comorbidity Survey

Seventy tetracycline-resistant Streptococcus pneumoniae were tested for the presence of tetracycline resistance genes, tetM and tetO, using a polymerase chain reaction (PCR) assay and DNA-DNA hybridization. Seven isolates representing five serotypes (12, 22, 6A, 19F and 23) carried the tetO gene. Five of the isolates were genetically unrelated as judged using pulsed field gel electrophoresis (PFGE) analysis. Two 19F isolates came from the same patient, carried both tetM and tetO genes and had the same PFGE pattern. The other 63 isolates carried only the tetM gene. DNA sequences from three of the tetO-carrying isolates were determined; they showed 91-95% nucleotide sequence identity over 300 nucleotides, and 93-95% amino acid sequence identity over 100 amino acids. The isolates carrying both tetO and tetM genes could transfer the tetM gene into both Enterococcus faecalis and S. pneumoniae recipients, but not the tetO gene. There was no detectable transfer of the tetO gene, by conjugation, from the other five isolates.     

Unusual antigenic and genetic characteristics of human respiratory syncytial viruses isolated in Cuba

The G protein of 23 strains of human respiratory syncytial virus isolated in Havana, Cuba, between October 1994 and January 1995 was analyzed at the antigenic and genetic level. All viruses reacted with 10 of 11 antibodies specific for the Long strain. Moreover, the G protein gene of the Cuban isolates had only five nucleotide differences from the sequence of the Long gene. The homogeneity of the Cuban isolates and their resemblance to an ancient strain, such as Long, are at odds with previous findings for viruses isolated in countries with a temperate climate and different socioeconomic status. The G proteins of three of four other viruses isolated in Havana 2 years later (1996) were also identical to those of the 1994-to-1995 isolates, and the fourth virus had a single extra nucleotide difference. This, again, is unusual, since no identical viruses had been isolated in different epidemics previously. The singular characteristics of the Cuban isolates reported here are discussed in terms of the epidemiological, climatic, and socioeconomic characteristics of Cuba.     

Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong

Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393-395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate's gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.     

Genetic variation of envelope gp90 gene of equine infectious anemia virus isolated from an experimentally infected horse

Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.     

Detection of West Nile virus by the polymerase chain reaction and analysis of nucleotide sequence variation

A polymerase chain reaction (PCR) assay was developed to rapidly detect and identify West Nile (WN) virus. The RNA from seven isolates of WN virus from six countries and four other flaviviruses (Kunjin, Japanese encephalitis, St. Louis encephalitis, and yellow fever viruses) was reverse-transcribed (RT) and amplified by PCR. The nucleotide sequences of the amplified products were determined by a rapid, automated DNA sequencing method. The WN virus RT/PCR assay detected the target gene segment of sequencing method. The WN virus RT/PCR assay detected the target gene segment of isolates from both the African-Middle Eastern group and the Indian group with a sensitivity of approximately 0.05 pg of viral RNA. Kunjin virus was the only other flavivirus tested that produced a band of the appropriate size. Five of seven WN virus isolates showed 92-98% homology in the nucleotide sequence of their PCR products. The sequence of one isolate was virtually identical to the published sequence of the Nigerian isolate (99.5% homology). No correlation was established between the degree of nucleotide homology, geographic location, time of isolation, or source of the isolates.     

Enhancing effect of L-carnitine on some abnormal signs induced by pentylenetetrazol

As indicated by their nucleotide sequences bovine viral diarrhea viruses (BVD-virus) are genetically quite heterogenous. Despite differences in the nucleotide sequence in the NS3 region, we found that the derived amino acid sequence of all viral strains analyzed was identical and differed from that of classical swine fever viruses, thus allowing differentiation between bovine and porcine pestiviruses. Remarkably, the BVD viral strains were isolated over a time period of some 40 years in Europe, North America and New Zealand. The nucleotide sequence of the 5' untranslated genome region of a BVD virus recently isolated in Switzerland from a calf with hemorrhagic syndrome differs from that of BVD viruses causing similar symptoms in a large epidemic outbreak in North America.     

Mycobacterium avium complex strains from AIDS patients belong to a homogeneous group ascribed by rRNA typing methods

16S rRNA RFLP analysis of Mycobacterium avium complex (MAC) strains isolated from 25 AIDS patients led to identification of seven ribotypes. The same ribotype was determined for strains from 19 patients with and without disseminated disease. When isolates representing the seven ribotypes were examined for their internal transcribed spacer (ITS) between the 16S and 23S rRNA gene nucleotide sequence, four different sequences, including a new ITS type, were recovered. All isolates with the most common ribotype belonged to the sequevar Mav-B. When MAC strains from AIDS patients were compared by ITS sequencing and ribotyping, a significant degree of homogeneity was observed. The discriminatory level reached with ribotyping might be useful for grouping isolates from different clinical sources.     

Evolutionary relationships among the serpins

The serpins are a widely distributed group of serine proteinase inhibitors found in plants, birds, mammals and viruses. Despite the great evolutionary divergence of these organisms, their serpins are highly conserved, both in sequence and structurally. Amino acid sequences were aligned by a combination of automatic algorithms and by consideration of conserved structural elements in those serpins for which crystal structures exist. The program HOMED was used which allowed the alignment of amino acids to be simultaneously converted into the equivalently aligned nucleotide sequences. The aligned amino acids were used as the basis for superposition of the four known three-dimensional structures for which coordinates are available and compared with an optimal three-dimensional superposition in order to estimate the reliability of the sequence alignment. Phylogenetic relationships implied by these nucleotide sequence alignments were determined by the method of maximum parsimony. The proposed gene tree suggested that as much diversity existed between the plant serpin and mammalian serpins as was present among mammalian serpins and provided further evidence that the architecture of serpin molecules is highly constrained.     

Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus

The nucleotide sequences of S1 glycoprotein genes of the Gray and JMK strains of avian infectious bronchitis virus (IBV) were determined and compared with published sequences for IBV. The IBV Gray and JMK strains had 99% nucleotide sequence similarity. The overall nucleotide sequence similarity of the Gray and JMK strains compared with other IBV strains was between 82.0% and 87.4%. The similarity of the predicted amino acid sequence for the S1 glycoproteins of the Gray and JMK strains was 98.8%. Six of the 10 differences in the amino acid sequence were found between residues 99 and 127, suggesting a possible role for that region in the tissue trophisms of the viruses. The S1 glycoprotein of the Gray and JMK strains had 79.5%-84.6% amino acid similarity with the published sequence of other IBV strains. Serine instead of phenylalanine was observed in the protease cleavage site between the S1 and S2 glycoprotein subunits for the Gray and JMK strains, which was similar to the published sequence for the Ark99 and SE17 strains. The significance of that amino acid change is not known. Based on the nucleotide sequence of the Gray and JMK strains, the BsmAI restriction enzyme was selected by computer analysis and was used in restriction fragment length polymorphism analysis to differentiate the two strains.     

Independent isolates of the emerging subgroup J avian leukosis virus derive from a common ancestor

A new subgroup of avian leukosis virus (ALV) that includes a unique env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.     

Characterization of a chromosomal gene encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal beta-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal beta-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand beta-lactamase production among phage group II staphylococci and the nature of the type B beta-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce beta-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B beta-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C beta-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of the mature exoenzyme form of type B S. aureus was determined. The deduced amino acid sequence revealed 3 residues in the leader peptide and 12 residues in the exoenzyme portion of the beta-lactamase that differ from the prototypic type A beta-lactamase sequence. These include the serine-to-asparagine change at residue 216 found in the kinetically similar type C enzyme, a threonine-to-lysine change at residue 128 close to the SDN loop (residues 130 to 132), and several substitutions not found in any of the other staphylococcal beta-lactamases. In summary, modern isolates of S. aureus typeable by group II phages produce type B or type C staphylococcal beta-lactamase. The type B gene resides on the chromosome and has a sequence that, when compared to the sequences of the other staphylococcal beta-lactamases, corresponds well with its kinetic properties.     

Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus

We report that cells refractory to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV) became susceptible when transfected with a chimeric aminopeptidase-N (APN) cDNA containing a canine domain between residues 643 and 841. This finding shows that APN recognition by these viruses is species related and associated with this C-terminal domain. The human/canine APN chimera was also able to confer susceptibility to the porcine transmissible gastroenteritis virus (TGEV), whereas its human/porcine homolog failed to confer susceptibility to CCV and FIPV. A good correlation was observed between the capacity of CCV, FIPV, and TGEV to recognize the different interspecies APN chimeras and their ability to infect cells derived from the relevant species. As an exception, TGEV was found to use a human/bovine APN chimera as a receptor although itself unable to replicate in bovine cells.