A three-generation family cluster with COVID-19 infection: should quarantine be prolonged?

OBJECTIVES: Families are a transmission route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because of the close contact. Monitoring of the viral load will be a valuable method to reduce the optimal number of quarantine days, especially in presymptomatic and symptomatic carriers of their households. The traditional three-generation families living together are seen frequently in East Asia, including in Taiwan. STUDY DESIGN: We report on a family cluster with six individuals infected with coronavirus disease in Taiwan. METHODS: The current public policy in Taiwan is quarantine for at least 14 days, based on the incubation period, or until the patient has tested negative three days in a row using the SARS-CoV-2 reverse transcription polymerase chain reaction. Details on the onset date of clinical symptoms, throat swab conversion, and course of disease were collected from medical records retrospectively. RESULTS: In the household of this three-generation Taiwanese family, the infection rate was 60%. The ratio of males to females was 4:2, and the age range was 11-85 years. The prevalence of asymptomatic disease was 33.3% (2/6). The longest throat swab conversion time was 37 days, and the estimated course of disease from symptoms to first conversion of throat swab was 59 days. CONCLUSIONS: Large families, including three-generation families in a single dwelling, should be monitored when the index case is found. Presymptomatic and symptomatic family members could be quarantined for an appropriate duration which, in our experience, is 2 months.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Isolation and characterization of a highly attenuated respiratory syncytial virus (RSV) vaccine candidate by mutagenesis of the incompletely attenuated RSV A2 ts-1 NG-1 mutant virus.

Ts-1, a temperature sensitive (ts) mutant of RSV, was previously derived from RSV A2 virus by mutagenesis with 5-fluorouracil (5-FU). Ts-1 was attenuated for adult volunteers and seropositive children but retained a low level of virulence in seronegative infant vaccinees as indicated by the occurrence of upper respiratory tract disease. Ts-1 NG-1, a more defective derivative of ts-1, was produced by mutagenesis of ts-1 with nitrosoguanidine. However, ts-1 NG-1 still retained a low level of virulence for the upper respiratory tract and showed some genetic instability in chimpanzees. With renewed interest in the goal of developing a live, attenuated RSV vaccine, we have now attempted to further attenuate ts-1 NG-1 by mutagenesis with 5-FU and 5-azacytidine. Four mutants that are phenotypically different from the ts-1 NG-1 parental virus were identified. Each of the four mutants was more restricted in replication in BALB/c mice compared with the ts-1 NG-1 parental virus. One of the ts-1 NG-1 derivatives, termed A-20-4, which showed the lowest (35 degrees C) in vitro shutoff temperature and which was also completely restricted in replication in BALB/c mice, was selected for further evaluation in seronegative chimpanzees. A-20-4 did not cause rhinorrhea in chimpanzees but induced detectable titers of serum RSV neutralizing antibodies in 2 of 4 chimpanzees. Apparent complete protection to subsequent challenge with wild-type RSV was observed in each of the four chimpanzees previously immunized with A-20-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of the NS3 serine proteinase of hepatitis C virus as determined by mutagenesis at the NS3/NS4A junction.

Hepatitis C virus (HCV) encodes a polyprotein that is processed to produce the structural and nonstructural proteins of the virus. Nonstructural protein 3 (NS3) is a serine proteinase that cleaves the polyprotein to release the NS4A, NS4B, NS5A, and NS5B proteins. To characterize the substrate specificity of NS3, we synthesized by in vitro translation the polyprotein NS2*-NS3-NS4*P that includes 70% of the NS2 protein, the complete NS3 protein, and 25% of the NS4 protein region attached to substance P, an epitope tag. We demonstrated that NS3 cleaves at the NS3/NS4A junction to release the NS4*P protein. Subsequently, we used this reaction to evaluate the importance of conserved amino acids that flank the NS3/NS4A junction. We replaced amino acids in the P6, P1, and P1' positions of the scissile bond of this junction using site-directed mutagenesis. When the P6 aspartic acid was changed to asparagine, lysine, or serine, NS3-mediated cleavage occurred. When threonine in the P1 position was replaced with other polar amino acids or with amino acids having aliphatic side chains, cleavage occurred, although it was not detected when arginine or tyrosine was present. Replacement of serine in the P1' position with other polar amino acids, with amino acids having aliphatic side chains, or with arginine resulted in NS3-mediated cleavage. Thus, since fewer amino acids in the P1 position supported cleavage than in the P6 or P1' positions, the P1 position of the scissile bond may play a more important role in defining the substrate specificity of the HCV NS3 proteinase.

Relevance of the guinea-pig necrotic inflammation footpad reaction to safety concerns with MDP-based adjuvants.

The guinea-pig necrotic inflammatory reaction described by Nagao and Tanaka was investigated to determine its possible relevance to tuberculin-positive individuals who may receive MDP-based adjuvants. The reaction was induced by a preparatory footpad injection of Mycobacterium tuberculosis in Freund's incomplete adjuvant (FIA) followed three weeks later by a provocative gluteal injection of muramyl dipeptide (MDP). Increased footpad swelling and necrosis occurred within 24 h. The reaction was also caused to a significantly lesser degree by a gluteal injection of the threonyl-MDP component of Syntex adjuvant SAF-m. Elicitation of the reaction in responsive animals was absolutely dependent on the presence of both FIA and M. tuberculosis at the reaction site. Animals injected in the right footpad with FIA plus M. tuberculosis and in the left footpad with M. tuberculosis alone responded to the provocative injection with necrosis in the right footpad only. The reaction therefore appears to have little potential relevance to the vaccination of tuberculin-positive humans.

Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis.

A cold-passaged RSV mutant, designated cp-RSV, which acquired host range mutations during 52 passages at low temperature in bovine tissue culture, was completely attenuated for seropositive adults and children but retained the capacity to cause upper respiratory disease in seronegative infants. We sought to introduce additional attenuating mutations, such as temperature-sensitive (ts) and small-plaque (sp) mutations, into the cp-RSV mutant, which is a ts+ virus, in order to generate a mutant which would be satisfactorily attenuated in seronegative infants and young children. Nine mutants of cp-RSV, which had acquired either the ts or small-plaque sp phenotype, were generated by chemical mutagenesis with 5-fluorouracil. The two ts mutants with the lowest in vitro shut-off temperature, namely the cpts-248 (38 degrees C) and cpts-530 (39 degrees C) mutants, were the most restricted of the nine cp-RSV mutant progeny tested for efficiency of replication in Balb/c mice. In seronegative chimpanzees, the cpts-248 mutant replicated fourfold less efficiently in the nasopharynx and caused significantly less rhinorrhoea than its cp-RSV parent. The cpts-248 mutant virus, like its cp-RSV parent, was 1000-fold restricted in replication in the trachea compared with wild-type RSV. Previously, another candidate RSV live attenuated vaccine strain, a mutant designated ts-1, exhibited some instability of its ts phenotype following replication in susceptible humans or chimpanzees. Hence, we sought cp-RSV ts progeny that exhibited a greater degree of stability of the ts phenotype than the prototype ts-1 mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1.

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

Efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model.

In the absence of an adequate small animal model for testing the efficacy of adenovirus-vectored respiratory syncytial virus (RSV) vaccines, a ferret model was established for this purpose. Recombinant adenovirus types 4, 5 and 7 expressing the RSV fusion glycoprotein (F), the attachment glycoprotein (G) or both F and G were constructed previously. These recombinants contain a deletion of a large portion of the E3 region of the respective adenovirus vector. In addition, an Ad7(E3+)F recombinant virus which contains an intact E3 region was constructed to assess whether E3 region functions might enhance vaccine immunogenicity. Evaluation of these viruses in the ferret model demonstrated that Ad4 and Ad5 recombinants, administered intranasally to ferrets, induce stronger seroresponses to RSV than do Ad7 recombinant viruses. Ad7(E3+)F did not show enhanced immunogenicity relative to E3-deleted recombinant viruses. However, measurement of RSV infectivity in nasal washes, following intranasal RSV challenge, showed that five different vaccination regimens, Ad7F/Ad4F, Ad7G/Ad4G, Ad7FG/Ad4FG, Ad4F/Ad7(E3+)F and Ad5F/Ad4F, protected ferrets from RSV infection in a dose-dependent manner.

Isolation and characterization of adenovirus-associated VA RNAs of human adenovirus type 7.

Two genes for virus-associated VA RNAs in the human adenovirus type 7 (Ad7) genome are compared to Ad2, Ad4, Ad5, simian VA RNAs, and pol III transcripts of Epstein-Barr virus. The newly identified VA RNA-encoding genes of Ad7 contain two relatively conserved intragenic promoter (elements A and B), the double-stranded RNA-dependent protein kinase (PK) active site-binding domain and transcriptional terminators. The conserved features extend to the VA RNA genes of simian and avian origin.

Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs.

High yielding adenovirus (Ad)-hepatitis B recombinant (Ad-Hep B) viruses were prepared by insertion of the hepatitis B surface antigen (HBsAg) gene into the early region 3 (E3 region) of Ad4 or Ad7 vectors containing intact or largely deleted E3 regions. Both E3-deleted and non-deleted recombinants produced about six- to eight-fold higher amounts of HBsAg than previously reported recombinants. These recombinant viruses were evaluated for immunogenicity in dogs which sustain abortive lung infections by Ad4 and Ad7. Recombinants containing E3 deletions elicited 10- to 12-fold stronger anti-HBs primary responses than previously evaluated low yield recombinants. Further immunizations with heterotypic Ad-Hep B recombinants induced substantial anti-HBs booster responses as well as anti-'a' epitope responses. In contrast, recombinant viruses containing intact E3 regions induced only weak or negligible anti-HBs responses, although such viruses induced strong antibody responses to the Ad vectors. The immunogenicity of high-yielding Ad recombinants correlated with temporal expression of HBsAg and thus the dog represents a valuable model for evaluation of immune responses to heterologous proteins that are expressed early and that do not require efficient DNA replication. Recombinants expressing HBsAg late in the infectious cycle require further testing in the fully permissive chimpanzee model. This study establishes that the E3-deleted high yield Ad4 and Ad7 recombinants represent promising live oral hepatitis B vaccine candidates.

Construction and characterization of adenovirus co-expressing hepatitis B virus surface antigen and interleukin-6.

Coexpression of biologically active interleukin 6 (IL-6), an immunoregulator, and hepatitis B virus surface antigen (HBsAg), an immunogen, was obtained using an adenovirus type 7 (Ad7) vector. Two recombinant adenoviruses (re-Ad) containing both the HBsAg and IL6 genes were constructed: one virus was capable of expressing IL6 with its signal peptide (spIL6) (Ad7::spIL6::HBsAg), and the second virus lacked this sequence (Ad7::IL6::HBsAg). A third recombinant contained only HBsAg (Ad7::HBsAg). All three Ad constructs were plaque purified and characterized in the A549 human lung cell line. The growth kinetics of the recombinants were similar to wild-type (wt) Ad7. The production and secretion of HBsAg (p24 and gp27) from cells infected with each re-Ad were at a level greater than 9 micrograms/10(6) cells by 118 h postinfection. Two IL-6 of approx. 24 and 27 kDa were produced and secreted into the culture medium from cells infected with Ad7::spIL6::HBsAg, and maximal accumulation occurred by 92 h p.i. at a level > 260 ng/10(6) cells. One cell-associated IL-6 of approx. 23 kDa was produced from cells infected with Ad7::IL6::HBsAg at a level > 12 ng/10(6) cells. Importantly, the Ad-produced IL-6 were determined to be biologically active by enhancing immunoglobulin production in lymphoblastoid cells. The co-production of IL-6 with HBsAg did not affect growth of these recombinant Ad, immunoreactivity of HBsAg, or the biological activity of IL-6 in tissue culture cells.

Colchicine disposition in human leukocytes after single and multiple oral administration.

Inasmuch as leukocytes were reported to be an active pharmacologic compartment, colchicine disposition was determined in plasma, granulocytes, and mononuclear cells in healthy volunteers after 1 mg oral single and multiple doses. After the single dose, maximal colchicine concentration was observed at 1 hour in plasma and 47 hours later in leukocytes. This delay was confirmed by the slow accumulation of colchicine by lymphocytes in culture. In the multiple-dose study, mean granulocyte colchicine concentration (20 to 53 ng/10(9) cells) were twofold higher than in mononuclear cells (9 to 24 ng/10(9) cells). Mean predicted colchicine multiple-dose granulocyte and mononuclear cell concentrations were 2.5-fold and ninefold higher, respectively, than those measured. After the last dose, colchicine decreased, with half-life values between 41 and 46 hours for leukocytes and 49 hours for plasma. This study validates leukocytes as a microcompartment whose kinetics correlates with colchicine biologic effects.

Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant.

Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques.

Cotton rats previously immunized with a chimeric RSV FG glycoprotein develop enhanced pulmonary pathology when infected with RSV, a phenomenon not encountered following immunization with vaccinia--RSV recombinants or RSV.

In studies conducted in the 1960s, children previously immunized with a formalin-inactivated respiratory syncytial virus (RSV) vaccine (FI-RSV) developed a greater incidence and severity of pulmonary disease during subsequent natural RSV infection than did controls. It was previously shown that cotton rats immunized with FI-RSV or immunoaffinity-purified fusion (F) glycoprotein developed enhanced pulmonary histopathology following intranasal challenge with RSV. In the present studies, various forms of immunization, including parenteral inoculation of an immunoaffinity-purified F glycoprotein or a chimeric FG glycoprotein produced in insect cells using a baculovirus vector (Bac-FG), intradermal infection with a vaccinia-F recombinant (Vac-F) or intranasal infection with an adenovirus-F recombinant (Ad-F) or RSV, were compared for immunogenicity, efficacy and ability to alter the host so that enhanced pulmonary histopathology developed during RSV infection 3 months after immunization. Immunization of cotton rats with F glycoprotein, Bac-FG, Vac-F, Ad-F or infection with RSV induced high levels of ELISA-F antibodies, but the antibodies induced by purified F glycoprotein of Bac-FG had low levels of neutralizing activity. Immunization with Vac-F or Ad-F, or infection with RSV induced a high level of resistance to pulmonary RSV replication, whereas animals immunized with Bac-FG or FI-RSV were only partially protected. Following RSV challenge, animals immunized with purified F glycoprotein or Bac-FG developed the highest levels of bronchiolar and alveolar histopathology, those immunized with FI-RSV had intermediate levels, and those immunized with Vac-F or RSV had histopathology scores at control levels. Ad-F immunized animals had elevated scores of bronchiolar but not alveolar histopathology; however, this finding was not reproducible. Passive transfer of pooled immune sera from animals infected with RSV or Vac-F and Vac-G was highly protective, whereas pooled sera from animals immunized with Bac-FG failed to protect the lungs against RSV challenge. Increased pulmonary histopathology was not observed in the passively immunized animals following RSV challenge, suggesting that the histopathology was mediated by RSV-specific T cells. These data indicate that subunit F glycoprotein or chimeric FG vaccines share with FI-RSV the properties of (i) induction of F antibodies with low neutralizing activity and (ii) enhancement of pulmonary histopathology during subsequent RSV infection. These observations confirm the need for caution in studies involving the administration of RSV subunit vaccines to seronegative humans.

Effects of purine nucleoside analogues with a cyclobutane ring and erythromycin A oxime derivatives on duck hepatitis B virus replication in vivo and in cell culture and HIV-1 in cell culture.

The effects on duck hepatitis B virus (DHBV) replication of specific analogues of two classes of chemical compounds not previously tested against hepadnaviruses are described. One is erythromycin A-9-methyloxime (EMO) and other oxime derivatives of erythromycin A, and the other is purine nucleoside analogues (cyclobut A and cyclobut G) with cyclobutane rings. Viral replication was assessed by measuring serum levels of DHBV DNA in infected ducklings and DHBV DNA in infected primary duck hepatocyte cultures. Administration of EMO 15 mg/kg of body weight IM to infected ducklings resulted in a rapid fall in DHBV DNA levels during therapy and a return to pretreatment levels after EMO administration was stopped. There was local toxicity at injection sites with muscle necrosis in some animals. When 100 mg/kg EMO was administered by gastric tube no such viral response was observed. The difference in virus response to EMO 15mg/kg IM and 100 mg/kg by gastric tube was not due to failure to achieve comparable blood and tissue levels of EMO administered by the different routes. The results suggest an indirect effect dependent on IM injection of EMO rather than a direct antiviral effect of the compound. Administration of cyclobut G or cyclobut A at 70 mg/kg IM led to a rapid reduction of DHBV DNA to undetectable levels in serum, and in only 1 of 4 animals did DHBV DNA became detectable again within 10 days after stopping the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of adenovirus type 4 and type 7 recombinant hepatitis B vaccines in dogs.

Recombinant hepatitis B virus vaccines based on adenovirus (Ad) vectors Ad4 and Ad7 have been prepared. However, immunogenicity testing of such vaccines in experimental animals is difficult because these human adenoviruses exhibit a highly restricted host range. In this study, the dog was evaluated as a model for screening Ad4- and Ad7-vectored vaccines. Intratracheal inoculation of dogs with Ad4 and Ad7 induced substantial type-specific humoral immune responses that were significantly higher than responses obtained following pharyngeal or oral inoculations. Inoculation of dogs with recombinant Ad7 and Ad4 vaccines expressing hepatitis B surface antigen (HBsAg) elicited large antibody responses to HBsAg (anti-HBs). Substantial secondary anti-HBs responses were produced upon sequential immunizations with heterotypic Ad7 and Ad4 recombinant vaccines. These data thus indicate that the dog is a useful model for evaluating immune responses to vaccines based on Ad4 and Ad7 vectors.

Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system.

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.