Growth Characteristics Of Virus Research Articles

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Correlation of SARS-CoV2 Viral Growth on Cultures, Ct Values, SARS-CoV-2 Variant and Vaccination Status in Asymptomatic, Pre-Symptomatic and Post-Infection Asymptomatic COVID Patients

Abstract Introduction/Objective COVID-19 large scale immunizations have helped control the infectious spread, but breakthrough infections are still observed and some of these patients remain asymptomatic. In this study, we compared the viral growth characteristics with vaccination status in asymptomatic patients. Methods/Case Report We searched our institutional laboratory database for asymptomatic patients infected with delta variant (November-December 2021, n=65) and omicron variant (December 2021, n=68), that were further classified as “truly asymptomatic”, “pre-symptomatic” and “post-infection asymptomatic” based on detailed chart reviews. The patients’ left-over nasopharyngeal swab samples’ cycle threshold values and the recovery of infectious virus in cell culture were compared. Results (if a Case Study enter NA) In the delta cohort, out of 65 asymptomatic patients, 36 were truly asymptomatic, 26 presymptomatic and 3 post infection asymptomatic patients. In the omicron cohort, out of 68 asymptomatic patients, 45 were truly asymptomatic and 23 were presymptomatic patients. The vaccination rate was 59% in truly asymptomatic (delta cohort=56%, omicron cohort=62%), 55% in presymptomatic (delta cohort=50%, omicron cohort=61%) and 67% in post-infection patients (delta cohort only). There was no significant difference in the Ct values of truly asymptomatic (mean Ct=21.08), presymptomatic (mean Ct=20.30) and post-infection asymptomatic patients (mean Ct=20.84) in the combined cohort (p=0.78) and in the delta (p=0.95) or omicron (p=0.45) subcohorts. The Ct values were not correlated with the vaccination status of the patients (vaccinated patients mean Ct=20.56, non-vaccinated patients mean Ct=20.16, p=0.68). Samples with positive viral growth on cultures had significantly lower Ct values (mean Ct= 17.16), compared to samples with negative viral culture results (mean Ct= 24.61) (p<0.0001). The viral growth on culture was not associated with the symptom status, SARS-CoV-2 variant type (delta vs omicron) and vaccination status of the patients in the delta and omicron subcohorts. Conclusion Our findings show that there is no significant correlation between vaccination status and viral loads and culture results in asymptomatic patients.

Isolation and Characterization of Five Severe Acute Respiratory Syndrome Coronavirus 2 Strains of Different Clades and Lineages Circulating in Eastern India.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts, and rapid emergence of new variants urge for the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first COVID-19 wave in Odisha, India. The viral isolates were adapted to in vitro cultures and further characterized to identify strain-specific variations in viral growth characteristics. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government-run vaccine drive in India. The major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host–virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.
 AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.
 MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.
 Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.
 CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

Cyanophage Propagation in the Freshwater Cyanobacterium Phormidium Is Constrained by Phosphorus Limitation and Enhanced by Elevated pCO2.

Intensification of human activities has led to changes in the availabilities of CO2 and nutrients in freshwater ecosystems, which may greatly alter the physiological status of phytoplankton. Viruses require hosts for their reproduction and shifts in phytoplankton host physiology through global environmental change may thus affect viral infections as well. Various studies have investigated the impacts of single environmental factors on phytoplankton virus propagation, yet little is known about the impacts of multiple factors, particularly in freshwater systems. We therefore tested the combined effects of phosphorus limitation and elevated pCO2 on the propagation of a cyanophage infecting a freshwater cyanobacterium. To this end, we cultured Phormidium in P-limited chemostats under ambient (400 μatm) and elevated (800 μatm) pCO2 at growth rates of 0.6, 0.3, and 0.05 d-1. Host C:P ratios generally increased with strengthened P-limitation and with elevated pCO2. Upon host steady state conditions, virus growth characteristics were obtained in separate infection assays where hosts were infected by the double-stranded DNA cyanophage PP. Severe P-limitation (host growth 0.05 d-1) led to a 85% decrease in cyanophage production rate and a 73% decrease in burst size compared to the 0.6 d-1 grown P-limited cultures. Elevated pCO2 induced a 96% increase in cyanophage production rate and a 57% increase in burst size, as well as an 85% shorter latent period as compared to ambient pCO2 at the different host growth rates. In addition, elevated pCO2 caused a decrease in the plaquing efficiency and an increase in the abortion percentage for the 0.05 d-1 P-limited treatment, while the plaquing efficiency increased for the 0.6 d-1 P-limited cultures. Together, our results demonstrate interactive effects of elevated pCO2 and P-limitation on cyanophage propagation, and show that viral propagation is generally constrained by P-limitation but enhanced with elevated pCO2. Our findings indicate that global change will likely have a severe impact on virus growth characteristics and thereby on the control of cyanobacterial hosts in freshwater ecosystems.

Isolation of cyanophage CrV infecting Cylindrospermopsis raciborskii and the influence of temperature and irradiance on CrV proliferation

A lytic virus that infects a European strain of the freshwater filamentous cyano-bacterium Cylindrospermopsis raciborskii was isolated from a lake in the Netherlands and partially characterised. With a genome size of 110 +/- 15 kb, an icosahedral capsid of 65 +/- 1 nm (n = 22) and a long non-contractile tail of 612 +/- 31 nm (n = 15), this dsDNA cyanophage CrV appears to belong to the Siphoviridae family. CrV was highly host specific, not infecting other filamentous cyano bacteria species isolated from the same lake, nor 4 Australian strains of C. raciborskii. The latent period of this cyanophage was 20-24 h. Varying the irradiance affected cyanophage-host inter actions: at low light (20 mu mol quanta m(-2) s(-1)) the latent period was 1.3 times longer compared with at mid light (90 mu mol quanta m-2 s(-1)); burst size at mid light was 332 CrV per lysed host cell, at low light it was halved (48%) and at high light (250 mu mol quanta m(-2) s(-1)) the burst size was further reduced to only 14% of that of mid light. Temperature also affected the virus growth characteristics: the CrV latent period at high temperature (30 degrees C) was reduced to just 11% (compared with a mid temperature of 22 degrees C), but still the burst size increased to 541 CrV per lysed host cell; at low temperature (15 degrees C) the latent period was prolonged 1.3-fold and the burst size was reduced to 43%. Our findings indicate that ecologically relevant environmental factors can affect the extent of viral lysis of C. raciborskii, advancing our understanding of the spread of this invasive cyanobacterium across Europe.

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems.

Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. Improved replication was observed for reassortants of the strictly avian IAV A/FPV/Rostock/34 (FPV, H7N1) containing the NS segment from A/Goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of A/Mallard/NL/12/2000 (MA, H7N3). The NS1 of GD and MA differ only in 8 aa positions. Here, we show that efficient replication of FPV-NSMA-derived mutants was linked to the presence of a single substitution (D74N) and more prominently to a triple substitution (P3S+R41K+D74N) in the NS1MA protein. The substitution(s) led to (i) increased virus titres, (ii) larger plaque sizes and (iii) increased levels and faster kinetics of viral mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth characteristics in avian cells. Furthermore, we show that an FPV mutant with N74 in the NS1 (already possessing S3+K41) is able to replicate and cause disease in mice, demonstrating a key role of NS1 in the adaptation of avian IAV to mammalian hosts. Our data suggest that (i) adaptation to mammalian hosts does not necessarily compromise replication in the natural (avian) host and (ii) very few genetic changes may pave the way for zoonotic transmission. The study reinforces the need for close surveillance and characterization of circulating avian IAV to identify genetic signatures that indicate a potential risk for efficient transmission of avian strains to mammalian hosts.

Shedding of Ebola Virus Surface Glycoprotein Is a Mechanism of Self-regulation of Cellular Cytotoxicity and Has a Direct Effect on Virus Infectivity.

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP.

Production of avian influenza virus vaccine using primary cell cultures generated from host organs

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2-10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.

Mutations at Alternative 5′ Splice Sites of M1 mRNA Negatively Affect Influenza A Virus Viability and Growth Rate

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.

Evolution of HIV-1 Isolates that Use a Novel Vif-Independent Mechanism to Resist Restriction by Human APOBEC3G

The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cDNA cytosines to uracils, leading to viral genomic strand G-to-A hypermutations. However, the HIV-1 Vif protein triggers APOBEC3G degradation, which helps to explain why this innate defense does not protect patients. The APOBEC3G-Vif interaction is a promising therapeutic target, but the benefit of the enabling of HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to find out whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants that resist restriction would emerge. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for more than five weeks, but replication was eventually detected in three cultures. Virus-growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient and that in fact, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of antiretrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication.

Single amino acid substitutions in the HSV-1 helicase protein that confer resistance to the helicase-primase inhibitor BAY 57-1293 are associated with increased or decreased virus growth characteristics in tissue culture

Two mutants (BAYr1 and BAYr2) that are 100-fold and >3000-fold resistant, respectively, to the helicase-primase inhibitor (HPI) BAY 57-1293 were derived from a plaque-pure parental strain, HSV-1 SC16 cl-2. BAYr1 has two substitutions in the HSV-1 helicase (UL5) protein (A4 to V; K356 to Q) and BAYr2 has one (G352 to R). It was shown reproducibly that BAYr1 grows to higher titres in tissue culture while BAYr2 grows more slowly than wild-type. Marker transfer experiments confirmed that K356Q and G352R are the drug-resistance mutations and that they are directly associated with differences in virus growth in tissue culture. When BAYr1 was tested in a murine infection model, this virus was shown to be fully pathogenic. We present evidence that single mutations close to a predicted functional domain of an essential HSV-1 replication enzyme (helicase) are associated with drug resistance and virus growth characteristics.

Differential Immune Responses and Pulmonary Pathophysiology Are Induced by Two Different Strains of Respiratory Syncytial Virus

In this study we performed comparisons of pulmonary responses between two different respiratory syncytial virus (RSV) antigenic subgroup A strains, A2 and Line 19. Line 19 strain induced significant dose-responsive airway hyperreactivity (AHR) in BALB/c mice at days 6 and 9 after infection, whereas the A2 strain induced no AHR at any dose. Histological examination indicated that A2 induced no goblet cell hyper/metaplasia, whereas the Line 19 induced goblet cell expansion and significant increases in gob5 and MUC5AC mRNA and protein levels in vivo. When examining cytokine responses, A2 strain induced significant interleukin (IL)-10 expression, whereas Line 19 strain induced significant IL-13 expression. When IL-13-/- mice were infected with Line 19 RSV, the AHR responses were abrogated along with gob5 gene expression. There was little difference in viral titer throughout the infection between the line 19- and A2-infected mice. However, the A2 strain grew to significantly higher titers than the Line 19 strain in HEp-2 cells in vitro. Thus, RSV Line 19-induced airway dysfunction does not correlate with viral load in vivo. These data demonstrate that different RSV strains of the same antigenic subgroup can elicit differential immune responses that impact the phenotypic expression of RSV-induced illness.

Use of a Murine Cytomegalovirus K181-Derived Bacterial Artificial Chromosome as a Vaccine Vector for Immunocontraception

Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181(Perth) strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors.

Clade B HIV-1 superinfection with wild-type virus after primary infection with drug-resistant clade B virus.

The immunological response to HIV-1 infection has been postulated to impede superinfection with a second virus; however, a few recent reports have documented cases of HIV-1 superinfection in humans either from different viral clades or from the same clade. To differentiate between co-infection and superinfection in a patient harboring a distinct wild-type HIV 4 months after primary infection with drug-resistant HIV. Detailed dye primer and clonal sequencing along with length polymorphism analysis was used to investigate the evolutionary linkage between viral populations sampled at different timepoints. After a set point viral load of -6000 copies HIV RNA/ml, the viral load jumped to 34 000 copies/ml at month 4 and, shortly after, to almost 200 000 copies/ml. At that time a second viral strain was first detected by dye primer sequencing of a pol fragment. These findings were confirmed by analysis of a 1300 bp gag-pol fragment and clonal sequencing and phylogenetic analysis of the V3 region. Length polymorphism analysis of the gp120 V4-V5 region showed that the second viral population was absent even as a minority population until month 4, when it was found to be the majority population, and the initial variant was present only as a minority. Both strains were subtype B. These data support intraclade HIV-1 superinfection by wild-type virus in the absence of antiretroviral therapy in a patient initially infected with drug-resistant HIV. The substantially different in-vivo viral growth characteristics observed illustrate the potential for superinfection to impact disease progression.

Araçatuba virus: a vaccinialike virus associated with infection in humans and cattle.

We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Effects of Initial Dose on Eastern Equine Encephalomyelitis Virus Dependent Mortality in Intrathoracically Inoculated <I>Culiseta melanura</I> (Diptera: Culicidae)

Viral growth characteristics that favor rapid and prodigious virion production may increase virus transmission but be detrimental to infected hosts. Several arboviruses, including eastern equine encephalomyelitis (EEE) virus, negatively affect the survival of their infected mosquito vectors. To test the hypothesis that the mosquito virulent properties of EEE virus are caused by the presence of intrinsic viral growth properties, we investigated the effects of infecting dose on the survival of intrathoracically inoculated Culiseta melanura (Coquillett). Daily survival of age-matched females inoculated with either a low initial dose of 10(1.5) plaque-forming units (PFUs) per mosquito or a high initial dose of 10(5.5) PFUs per mosquito was monitored for 8 wk. Compared with diluent inoculated controls, mosquitoes from both dosage groups displayed highly significant decreases in survival. No significant differences in daily survival were detected between the two infected groups. Virus production within inoculated mosquitoes was assessed by sampling mosquitoes every 12 h for 96 h after inoculation. Rapid virus amplification occurred in both dosage groups, and by 24 h after exposure the mean viral loads in mosquitoes inoculated with the low dose were comparable to those inoculated with the high dose. Likewise, although detectable virions appeared sooner in the saliva of high dosage mosquitoes, by 72 h after inoculation no significant differences in virus transmission were detected between the two exposure groups. These results indicate that the virulence of EEE virus for its enzootic North American mosquito vector is not dosage dependent and likely reflects the inherent growth properties of this virus within infected mosquitoes.

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.