In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus.

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

Adsorption of Cholera and Heat-Labile Escherichia coli Enterotoxins by Various Adsorbents: an In Vitro Study.

A variety of common inorganic adsorbents representing aluminas, zeolites, phyllosilicate clays, silica, and carbon were compared for their abilities to adsorb cholera toxin (CT) and heat-labile (LT) Escherichia coli enterotoxin. An appropriate assay system for the enterotoxins was developed using the Y-1 mouse-adrenal-tumor cell line, End points were determined by counting the number of rounded (cytotonic) cells at the relevant dilution. The adsorption varied between 177.0 × 106 and 109.6 × 102 CYTU (cytotonic titer unit) for CT with charcoal and boehmite respectively, and between 60.7 × 104 and 180.4 × 101 CYTU for LT with charcoal and boehmite respectively. Several of the other materials adsorbed CT and LT well, particularly attapulgite and sodium bentonite. The tightness of CT and LT binding to sodium bentonite and charcoal was determined by washing the adsorbent-enterotoxin pellets. Both toxins were strongly adsorbed, with dissociation of only 46.3 × 10° CYTU (<0.01 %) of the bound CT from sodium bentonite and 18.0× 101 CYTU (0.06%) of the bound LT from charcoal. The clay and charcoal pellets were assayed for their cytotonicity. Most of the activity of the adsorbed enterotoxins was lost: 93.1 and 89.6% for CT with sodium bentonite and charcoal, respectively, and 93.8 and 85.9% for LT with sodium bentonite and charcoal, respectively. The effect of dietary protein (casein) in enterotoxin adsorption by clay was also investigated. One percent casein (when adsorbed to sodium bentonite clay) completely blocked the adsorption of CT. When this protein-clay complex was treated with enzymes present in pancreatin, the digestive effect on the casein was sufficient to permit the adsorption of 137.6 × 101 CYTU of CT, although most of the blocking effect of casein remained. Further in vitro studies are needed to model the stomach, pancreatic, and intestinal digestive systems for determining if dietary proteins can block CT adsorption by clay in vivo. These results extend and support previously published data, obtained experimentally in rabbit and rat intestinal loops and from studies of children suffering spontaneous diarrhea, on the beneficial role of clays and other inorganic adsorbents in controlling enterotoxin activity.

A major rearrangement of the VP6 gene of a strain of rotavirus provides replication advantage.

During coinfection of BSC-1 cells with bovine rotavirus B223 and human rotavirus 69M and subsequent serial passages at low multiplicity of infection (0.1 m.o.i.), a reassortant virus (BMR) with a rearranged VP6 gene became the predominant strain. At passage 24 virus extracted from 50 of 51 plaques (98%) contained the rearranged gene 6, which had been first observed in passage 19. The analyses of the clones obtained from passages before the appearance of the rearranged VP6 gene (passage 15) and after (passage 20) indicated that the B223 VP6 gene was the origin of the rearranged VP6 gene. To test whether the rearranged VP6 gene was responsible for the selection advantage observed, reassortant C11 was generated with BMR and WA rotavirus, containing the rearranged VP6 gene and the other 10 genes from WA. Coinfection of WA rotavirus and reassortant C11 and subsequent serial passages at low m.o.i. resulted in 100% of virus from clones extracted at passage 18 being identical to reassortant C11; demonstrating that the rearranged VP6 gene was once again selected over the normal VP6 gene. The selection advantage of the rearranged VP6 gene could not be explained by comparison of the growth curves of the viruses, as there was no significant difference between the growth cycles of rotavirus B223 and reassortant BMR, nor between rotavirus Wa and reassortant C11. However, the plaque and electropherotype analysis at passage 1 of Wa and C11 coinfection revealed that 85% of the progeny viruses contained the rearranged gene 6. These data show that the gene 6 rearrangement resulted in selection of the relevant reassortant, possibly by suppression of competitive strains, and may indicate a new mechanism for the evolution of rotavirus.

An unusual group-A rotavirus associated with an epidemic of diarrhea among three-month-old calves.

During the 1992 and 1993 breeding seasons, epidemics of diarrhea among calves approximately 3 months old in a cow/calf operation were reported. Rotavirus was determined to be the probable cause, but because rotavirus typically affects younger calves, further investigations were conducted to determine the characteristics of the virus. Virus isolated from the feces of 1 affected calf was found to be antigenically distinct from the vaccine strain used. The primary water source was a slough, and rapid spread of infection may have been a result of fecal contamination of the slough. In both years, the epidemic began shortly after migrating cattle egrets arrived in the district.

Studies on the influence of the VP7 gene on rotavirus replication.

In a previous study using reassortant viruses, bovine rotavirus B223 VP7 protein enhanced the neutralization titers of some VP4 specific cross-reactive monoclonal antibodies (MAbs) (Xu and Woode, Virology, 1993). In this report, the influence of the B223 VP7 gene on the growth of reassortants was studied. The growth curves of three B223/69M reassortants with or without the B223 VP7 gene were compared. Reassortant M8, which has B223 VP7 on the genetic background of 69M, replicated as fast and to a similar titer as B223 and better than 69M. In contrast, the growth of reassortants B9, which has 69M VP7 on the B223 background, and of M46, which has B223 VP4 and VP6 on the 69M background, were poorer than B223. As B223 VP7 appeared to provide replication advantages, B223 was cultured as co-infections with each of the following viruses: Wa, 69M, H-2, SA11-4F, and NCDV followed by 20 subpassages. For co-infections with Wa, 69M, H-2, and SA11-4F, B223 VP7 gene was selected, in contrast to the B223/NCDV co-infection, when NCDV VP7 gene was selected. Other genes were also selected non-randomly, but varied among the five co-infection pairs. These data suggest in particular that B223 or NCDV VP7 protein can provide replication advantages to reassortant viruses, possibly by modifying VP4 to make it adsorb more efficiently to the cell receptors.

Studies on the role of VP4 of G serotype 10 rotavirus (B223) in the induction of the heterologous immune response in calves.

In a previous study, convalescent antisera from gnotobiotic calves (GC) infected with the G10 serotype bovine rotavirus (B223) were found to neutralize a number of rotaviruses representing G1-G6 and G8-G10 serotypes, except for a G8 serotype 69M (Z. Xu et al., Vet. Microbiol., 1993). In order to determine the immunodominant antigen, a panel of reassortants between B223 and 69M was generated and tested with the B223 GC antisera. It was found that the antisera neutralized the infectivities of the reassortants containing VP7 of B223 at titers similar to those with the parental virus B223, whereas the neutralization titers with the reassortants containing VP4 but not VP7 from B223 were significantly lower. These titers were close to the heterologous titers obtained with B641 (a G6 serotype rotavirus) against which B223 induced protective immunity. In contrast, the reassortants with both VP4 and VP7 from 69M were not neutralized by the B223 GC antisera. This indicates that the immunodominant neutralizing antigen during the primary immune response in calves is VP7 and the heterologous response is probably directed at VP4. Evidence was obtained through a cross-reactive B223 VP4 monoclonal antibody (MAB), B223-N6, that a shared epitope exists among B223, B641, and various G serotype rotaviruses. These data support the conclusion that VP4 is an important antigen for G10 as well as G6 rotavirus immunity. The B223/69M reassortants were also assayed with two cross-reactive VP4 MABs B223-N6 and 2G4. B223 VP7 was found to enhance the neutralization titers of the MABs with rotaviruses containing either homologous (B223) VP4 or the heterologous (69M) VP4.

Immunodominant neutralizing antigens depend on the virus strain during a primary immune response in calves to bovine rotaviruses.

Sera obtained from gnotobiotic calves (GC antisera) infected with bovine rotavirus strain NCDV or B223 from a previous study (Woode et al., 1987), which have different G (G6 and G10 respectively) and P serotypes, were compared for their neutralization (NT) properties to a number of human and animal rotaviruses (representing G serotype 1-6, 8-10). Two distinct patterns of neutralization were identified from these GC antisera. Of all the serotypes tested, NCDV GC antisera neutralized only B641 to a relatively high titer compared with the homologous titer, implying a narrow pattern of NT response. Analysis with reassortants indicated that the response was primarily to VP4. In contrast, B223 GC antisera neutralized most of the G serotypes tested to titers within 3-7 fold of the homologous titer, demonstrating a broad pattern of NT response. In the earlier study B223 was shown to induce a heterotypic protection against bovine rotavirus B641 (G serotype 6), and the serologic data obtained from this study indicates that a B223 vaccine might provide broad protection against several different serotypes of human and animal rotaviruses.

Both surface proteins (VP4 and VP7) of an asymptomatic neonatal rotavirus strain (I321) have high levels of sequence identity with the homologous proteins of a serotype 10 bovine rotavirus.

The nucleotide sequence of genes 4 and 9, encoding the outer capsid proteins VP4 and VP7 of a serotype 10 tissue culture-adapted strain, I321, representative of asymptomatic neonatal rotaviruses isolated from neonates in Bangalore, India, were determined. Comparison of nucleotide and deduced amino acid sequences of I321 VP4 and VP7 with previously published sequences of various serotypes revealed that both genes were highly homologous to the respective genes of serotype 10 bovine rotavirus, B223. The VP4 of I321 represents a new human P serotype and the I321 and related strains represent the first description of neonatal rotaviruses that appear to derive both surface proteins from an animal rotavirus.

The outer capsid protein VP4 of equine rotavirus strain H-2 represents a unique VP4 type by amino acid sequence analysis.

The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population.

Two-way cross-neutralization mediated by a shared P (VP4) serotype between bovine rotavirus strains with distinct G (VP7) serotypes.

Among bovine rotavirus strains, there are three G serotypes (G6, G8, and G10) and three P (VP4) serotypes (PB1, PB2, and PB3, which are defined on the basis of strains NCDV, UK, and B223, respectively). Plaque reduction neutralization assays with hyperimmune antisera disclosed two-way antigenic relationships of strain KN-4 with strain KK-3 (G10, PB3) as well as with strains NCDV (G6, PB1) and 0510 (G6, PB2). Neutralization assays with monoclonal antibodies specific for G6, G10, and PB3 revealed that KK-3 and KN-4 had the same P serotype (PB3) but that neither G6- nor G10-specific monoclonal antibody neutralized KN-4. Comparison of the VP7 gene sequence of KN-4 with those of other bovine rotavirus strains indicated that KN-4 was more similar to G6 bovine strains than to KK-3 and other G10 strains, suggesting that the G serotype of KN-4 was G6. From these results, we concluded that the two-way cross-neutralization between KN-4 and NCDV or 0510 was mediated by shared G6 serotype specificity, whereas the two-way cross-neutralization between KN-4 and KK-3 was mediated by shared P serotype specificity (PB3). Thus, KN-4 and KK-3 represent the first reported example of a two-way antigenic relationship mediated only by the P serotype. This article emphasizes the need for adopting a binary serotyping system and development of reagents which will enable classification of rotaviruses based on their G and P serotype specificities.

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.

Myoelectric activity of the small intestine in enterotoxin-induced diarrhea of calves.

Electrodes were surgically implanted at 15-cm intervals in the jejunum and ileum of 4 healthy neonatal calves so that myoelectric activity could be recorded on 2 consecutive days. On the first day, each calf received a control treatment, and myoelectric activity was recorded for 340 minutes. Phase I was recorded for a mean of 175.8 +/- 22.8 minutes (51.5%), phase II for 124 +/- 27.4 minutes (36.5%), and phase III for 40.3 +/- 6 minutes (11.9%). On the second day, each calf was treated with approximately 200 micrograms of heat-stable enterotoxin (STa) of Escherichia coli orally. All calves developed diarrhea after the administration of STa. Phase I was recorded for a mean of 92.5 +/- 42.3 minutes (27.2%), phase II for 227.3 +/- 52.5 minutes (66.9%), and phase III for 20.3 +/- 11.4 minutes (6.0%). Increase in phase II and decrease in phases I and III after STa administration were significant (P less than 0.05). Duration of the migrating myoelectric complex was longer after STa administration (median, 64 minutes), compared with the control treatment (median, 54 minutes). Minute rhythms, recorded on the day of toxin administration, ranged from 49 to 153 minutes. There was no difference between the number of migrating action potential complexes on the control days (range, 1 to 10), compared with those on treatment days (range, 1 to 14). These findings are suggestive that enterotoxin-induced diarrhea of calves is accompanied by increased total spiking activity and minute rhythms in the distal portion of the jejunum and ileum.

Comparative amino acid sequence analysis of VP4 for VP7 serotype 6 bovine rotavirus strains NCDV, B641, and UK.

In a previous study (S. Zheng, G. N. Woode, D. R. Melendy, and R. F. Ramig, J. Clin. Microbiol. 27:1939-1945, 1989), it was predicted that the VP7 serotype 6 bovine rotavirus strains NCDV and B641 do not share antigenically similar VP4s. In this study, gene 4 and the VP7 gene of B641 were sequenced, and the amino acid sequences were deduced and compared with those of NCDV and bovine rotavirus strain UK. Amino acid sequence homology in VP7 between the three strains was greater than 94%, confirming their relationship as VP7 serotype 6 viruses. VP4 of B641 showed amino acid homology to UK of 94% but only 73% homology to NCDV. Sequence comparison of a variable region of VP8 demonstrated amino acid homology of 53% between B641 and NCDV, whereas B641 and UK were 89% homologous in this region. These results confirm the earlier prediction that although the same serotype by VP7 reactivity, B641 and NCDV represent different VP4 serotypes. This difference in VP4 may have contributed to the lack of homotypic protection observed in calves, implicating VP4 as an important antigen in the active immune response to rotavirus infection in bovines.

The shedding of group A rotavirus antigen in a newly established closed specific pathogen-free swine herd.

A longitudinal study was undertaken in a newly established specific pathogen-free (SPF) swine herd to determine the dynamics of rotavirus antigen shedding in a closed swine facility. Pregnant SPF gilts which populated the herd, and their offspring, were monitored weekly for three consecutive lactations. Fecal samples were assayed for the presence of group-specific viral antigen by a solid phase immunoassay (ELISA). Results indicate that in the week prior to farrow, 35% of samples from gilts/sows contained rotavirus antigen. During nursing, 37% of the gilts'/sows' fecal samples also contained virus antigen. Over the course of three farrowings, every gilt/sow in the herd excreted virus antigen. Virus antigen was present in 25% of the samples tested from nursing pigs and in 70% of the samples tested from pigs in the postnursing period; 95% of the litters excreted virus antigen either while nursing or postweaning. Seasonal incidence in virus antigen excretion was noted with proportionally more suckling pigs virus antigen-positive in summer and proportionally more sows/gilts positive during winter. Diarrhea occurred only rarely in the sampled population. Although piglets shed rotavirus subclinically, ELISA positive feces from piglets of each lactation caused severe disease when fed to neonatal gnotobiotic pigs. Electropherotyping of these passaged viruses indicated minor variation in RNA banding patterns over time.

A longitudinal study of rotavirus antibody titers in swine in a closed specific pathogen-free herd.

In a newly established closed specific pathogen-free (SPF) swine herd, gilt/sow suckling and weaned pig rotavirus specific antibody titers were followed for three lactations by enzyme-linked immunosorbent assay (ELISA) to gain insight into the dynamics of herd antibody titers to group A rotavirus. Among gilts/sows, serum antirotavirus IgG titers increased during each lactation with a subsequent drop in titer between farrowings. Serum antirotavirus IgM titers declined during each lactation and with subsequent parity. Serum antirotavirus IgA titers remained constant during lactations and among parities. In colostrum and milk, antirotavirus IgA antibody was abundant. Differences in titer were not noticed between gilts and second litter sows but third litter sows had significantly higher titers than the first two groups. Antirotavirus IgG was high in colostrum but nearly nonexistent in milk. This titer did not vary significantly within or among parities. There was a linear regression in the titers of baby pig serum antirotavirus IgG from the post colostral sample through to seven weeks old, after which titer began to increase. No difference in baby pig serum antirotavirus IgG was noted among the three litters. Serum antirotavirus IgA and IgM were undetectable in baby pig sera after 2-3 weeks of age. Coproantibody to rotavirus was sporadically present in pig feces for 2-3 weeks after birth with highest titers in the IgA fraction. We conclude that although it is probable that age resistance of pigs to rotavirus diarrhea occurs, humoral immunity as measured by ELISA rotavirus antibody titers may not be intimately involved in virus clearance since in our studies baby pigs passively received large amounts of antibody but still excreted pathogenic virus. The finding of increasing levels of serum antirotavirus IgG in gilt/sow serum suggest that exposure to antigen of dams occur without significant increases in antirotavirus IgG titers in either colostrum, milk, or baby pig serum.

Analysis of serotypes and electropherotypes of equine rotaviruses isolated in the United States.

Equine group A rotaviruses isolated over a 10-year period in New York State, New Jersey, Kentucky, and Texas were compared serotypically and electropherotypically. All isolates were determined to be serotype 3 by reaction with hyperimmune antiserum to the serotype 3 H-2 strain of equine rotavirus. All displayed RNA electrophoretic migration patterns related to that of the H-2 strain but distinct from that of serotype 5 strain H-1. A serologic survey of 184 mares in Kentucky, which was done to determine the incidence of H-1 and H-2 infections, showed geometric mean serum neutralizing titers to the H-2 strain of equine rotavirus to be significantly higher than those to the H-1 strain. These data suggest that the serotype 3 H-2 strain is the dominant equine rotavirus in Kentucky and perhaps elsewhere in the United States. We were unable to produce confirmational evidence that the H-1 strain occurs as a natural infection in the United States.

Comparative studies of the antigenic polypeptide species VP4, VP6, and VP7 of three strains of bovine rotavirus.

Three bovine rotavirus strains belonging to two distinct serotype groups, serotype 6 (NCDV and B641) and B223, distinct from the other six mammalian rotavirus serotypes but not yet assigned to a serotype group, were compared with each other and with canine rotavirus (K9, serotype 3) by studying the properties of their cognate polypeptide species VP4, VP6, and VP7. The three viruses showed distinct differences in the polyacrylamide gel electrophoretic migration rates of protein species VP4 and VP7, with minor differences in VP6. Differences were also observed among the migration patterns of genome segments 4, 6, and the 7-8-9 triplet, which encode VP4, VP6, and VP7, respectively. Monoclonal antibodies (MAbs) to B223, which were directed against VP4 or VP7, showed homologous specificity for neutralization and immunofluorescence (IF), although one MAb reactive with VP4 also reacted by IF and by immunoprecipitation (IP) with all four viruses and weakly neutralized B641 and K9. This MAb may react with the epitope responsible for the B223-induced one-way neutralizing and protection response of calves against B641 observed in earlier studies. MAbs reactive with VP6 by IP showed enzyme-linked immunosorbent assay and IF reactivity with all three bovine viruses and the canine virus. The two serotype 6 viruses could be distinguished by the two B641 MAbs, B641-N2b reacting by neutralization and IF with both viruses and B641-N1 reacting with B641 and the serotype 3 canine rotavirus but not with NCDV. One nonneutralizing B641 MAb reacted by IP and IF with VP7 of all four rotaviruses examined, and one B223 MAb neutralized B223 and, to low titer, B641 and K9 although reacting by IP and IF with all four viruses. Three MAb-resistant mutants were selected by passage of B223 in the presence of one of three selected B223 MAbs at concentrations which only neutralized approximately 90% of the infectious virions. The resulting mutants were 100% resistant to neutralization with their respective MAb but remained neutralizable by the same selection of MAbs as the parent B223 virus.

Cultivation and partial characterization of bovine astrovirus.

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.