Persistent Carrier State Research Articles

New Approaches to Tackling Intractable Issues in Infectious Disease.

Despite major progress in the last several decades in reducing the public and animal health burden of infectious disease a number of issues remain to be resolved and which have thus far been regarded as intractable. These include (i) the persistent carrier state in individuals convalescent from typhoid and typhoid-like infections, (ii) the increasing prevalence of multi-antibiotic resistance in enteric pathogens, much of which is mediated by self-transmissible plasmids, and (iii) parasite infections which are difficult to control by vaccination and where resistance to chemotherapeutics is also increasing. The author describes very recent work carried out by his group to look at resolving these problems in new and imaginative ways.

Short communication: Retrospective analysis of obligatory testing results for Equine virus arteritis reveals a decrease of its seroprevalence in stallions used for artificial insemination

Equine viral arteritis (EVA) can induce a persistent carrier state in stallions which then shed the virus via semen. About 30 years ago, obligatory EVA testing of stallions used for artificial insemination (AI) was implemented in the European Union. Information on the efficacy of these regulations on the prevalence of EVA in stallions are not yet available. Therefore, we retrospectively analyzed results of serological and virus antigen testing for EVA in sires of different age and breed referred to Vetmeduni Vienna for semen preservation or veterinary diagnostic procedures between 2001 and 2021. For analysis, stallions were grouped by age (1–5, 6–8, 9–12, >12 years) and breed. The EVA antibody titer was determined by serum neutralization test and semen was analyzed for EVA virus by PCR and/or virus isolation test. Of 308 stallions tested, 14.9% (n = 46) were EVA seropositive and in 12 stallions EVA virus was detected in semen (26% of seropositive stallions). The incidence of seropositive stallions decreased over time (P < 0.05, χ2 test). Differences in the seroprevalence of EVA antibodies existed among stallion age groups (P < 0.01, Fisher’s test) with the highest percentage of seropositive stallions being > 12 years old (43.5%). The EVA antibody titer increased with age (P < 0.01, Kruskal-Wallis test), potentially reflecting repeated virus challenge. In conclusion, analysis of monitoring results revealed a decrease of EVA seroprevalence and virus shedding in a European sire population. As monitoring for EVA was the only measure implemented Europe-wide, testing might be a major contributor to this development.

Monitoring for Equine Viral Arteritis has decreased its prevalence in European breeding sires

Equine viral arteritis (EVA) can induce a persistent carrier state in stallions and shedding of the virus with semen can spread EVA and cause abortions in seronegative pregnant mares. In the early 1990s, obligatory testing for EVA of stallions used for artificial insemination was implemented within the European Union (Council Directive 92/65/EEC). In addition, vaccination programs for young stallions and exclusion of carrier stallions from certain breed registry events were implemented in Warmblood sport horses. Information on the efficacy of these actions on the prevalence of EVA in reproductively active stallions are not yet available in Europe. This study retrospectively analyzed results of serological and virus antigen testing for EVA in sires (n=308, one sample set per sire, EVA-vaccination status unknown) of different age and breed referred to the Centre for Artificial Insemination of Vetmeduni Vienna for semen preservation between 2001 and 2021. For analysis, stallions were grouped by age (1-3, 4-10, 11-16, >16 years) and breed. The EVA antibody titer was determined by serum neutralization test and semen was analyzed for EVA virus by PCR and/or virus isolation test. Statistical analysis was performed by chi2-test, Kruskal-Wallis test or Mann-Whitney U-test using the IBM SPSS 28.0 statistics software. Of 308 stallions tested, 14.9% (n=46) were EVA seropositive and in 12 stallions EVAvirus was detected in semen (26% of seropositive and 3.8% of all tested stallions, respectively). The incidence of seropositive stallions (22% of sires tested during 2001-05 to 4.1% during 2016-21) and of stallions shedding EVA virus via semen (8.5% of all serologically tested stallions 2001-05 to 1.4% during 2016-21) decreased over time (p<0.05). No difference among breeds with regard to occurrence of seropositive stallions was detected. There was a tendency towards more Standardbred and Lipizzaner stallions (14% and 13%, respectively) shedding EVA virus in semen compared to all other breeds (<3%, p=0.074 among groups). Differences in the seroprevalence of EVA antibodies existed among stallions from different age groups (p<0.01) with the highest percentage of seropositive stallions in 11-16 year-stallions (45.7%). No differences among age groups with regard to virus shedding existed. The EVA antibody titer increased with age (p<0.01), reflecting a higher possibility of repeated virus challenge in older stallions. Antibody titer was not associated with carrier state. In conclusion, monitoring of stallions for EVA in combination with vaccination programs resulted in a decrease of the prevalence of seropositive and virus-shedding stallions during the past 20 years in a European stallion population and can therefore be considered successful.

Investigations into the carrier-state of Theileria sp. (buffalo) in cattle.

The Theileria are apicomplexan parasites transmitted by ticks to vertebrate hosts. Most Theileria species exhibit some form of host or vector specificity, since under endemic conditions only a limited number of tick species act as vectors and not all vertebrate hosts are able to maintain a persistent carrier state. Data for Theileria sp. (buffalo) suggest host specificity for African buffalo (Syncerus caffer). However, T. sp. (buffalo) infections in cattle co-grazing with African buffalo have been reported in Kenya and schizonts were cultured from these infected cattle, raising questions regarding host specificity. A Corridor disease outbreak in 2013 on a ranch in South Africa where cattle co-grazed with Theileria parva and T. sp. (buffalo) infected buffalo presented the opportunity to investigate the possible carrier-state of T. sp. (buffalo) in cattle using real-time PCR analysis. Almost all buffalo (n = 19, 95%) were infected with T. sp. (buffalo) and showed CP values (22-20) indicative of high parasitemia similar to that observed for buffalo in endemic areas. Conversely, only ~14-27% cattle (n = 69, 100, 96) were positive with CP values (31-40) suggesting low parasitemia and a carrier state epidemiology different from African buffalo. Long term monitoring of T. sp. (buffalo) positive cattle showed that most cattle lost their parasitemia or presented fluctuating parasitemia around the PCR assay detection limit. A single splenectomized animal showed a persistent carrier state. The general trends and epidemiology observed in cattle infected with T. sp. (buffalo) are similar to that seen for buffalo-adapted T. parva, for which a defined carrier state in cattle has not yet been proven. The study suggests that cattle may be infected by T. sp. (buffalo) but are not definitive hosts that play an important part in the epidemiology of this parasite.

Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera.

The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.

Characterization of simian T-cell leukemia virus type 1 in naturally infected Japanese macaques as a model of HTLV-1 infection

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed.ResultsWe identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1+ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration.ConclusionsSTLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals.

Induction of a broad spectrum of inflammation-related genes by Coxsackievirus B3 requires Interleukin-1 signaling

Coxsackievirus B3 (CVB3) is a major cause of acute and chronic forms of myocarditis. Previously, direct viral injury and post-infectious autoimmune response were suspected as main pathogenetic mechanisms. However, induction of pro-inflammatory cytokines may be crucial for pathogenesis in spite of host protein shut off caused by CVB3 replication. We investigated the global expression profile of pro-inflammatory genes induced by acute and persistent (carrier state) CVB3 infection in human fibroblast cell cultures with DNA microarrays, quantitative RT-PCR and ELISA. Rapid induction of a typical spectrum of about 30 inflammation-related genes (e.g., PTGS2, CCL2, IL-1β, IL-6, IL-8, CSF2, MMP-1, MMP-3, and MMP-15) suggested an essential, autocrine role of IL-1. This hypothesis was confirmed by over-expression of IL-1RI, which resulted in a cytokine response upon CVB3 infection in HEK 293 cells otherwise refractory to CVB3-caused gene expression. Blocking IL-1 receptor type I (IL-1RI)-signaling during CVB3 infection with an IL-1 receptor antagonist (IL-1ra) as well as knockdown of IL-1RI using siRNA abrogated cytokine response in human fibroblasts. Both IL-1α and IL-1β are relevant for the induction of inflammation-related genes during CVB3 infection as shown by neutralization experiments. Paracrine effects of IL-1 on the subset of non-infected cells in carrier state infected fibroblast cultures enhanced induction of inflammation-related genes. A broad spectrum of inflammatory cytokines was induced by CVB3 replication via a pathway that requires IL-1 signaling. Our results suggest that IL-1ra may be used as a therapeutic agent to limit inflammation and tissue destruction in myocarditis.

Age-structured dynamic, stochastic and mechanistic simulation model of Salmonella Dublin infection within dairy herds

In the demand for a decision support tool to guide farmers wanting to control Salmonella Dublin (S. Dublin) in Danish dairy herds, we developed an age-structured stochastic, mechanistic and dynamic simulation model of S. Dublin in dairy herds, which incorporated six age groups (neonatal, preweaned calves, weaned calves, growing heifers, breeding heifers and cows) and five infection states (susceptible, acutely infected, carrier, super shedder and resistant). The model simulated population and infection dynamics over a period of 10 years in weekly time steps as: 1) population sizes of each of the six age-groups; 2) S. Dublin incidence and number of animals in each infection state; and 3) S. Dublin related morbidity and mortality in the acutely infected animals. The effects of introducing one infectious heifer on the risk of spread of S. Dublin within the herd and on the duration of infection were estimated through 1000 simulation iterations for 48 scenarios. The scenarios covered all combinations of three herd sizes (70, 200 and 400 cows), four hygiene levels indicating infectious contact parameters, and four herd susceptibility levels indicating different susceptibility parameters for the individual animals in each of the six age groups in the herd.The hygiene level was highly influential on the probability that the infection spread within the herd, duration of infection and epidemic size. The herd susceptibility level was also influential, but not likely to provide sufficient prevention and control of infection on its own. Herd size did not affect the probability of infection spread upon exposure, but the larger the herd the more important were management and housing practices that improve hygiene and reduce susceptibility to shorten durations of infection in the herd and to increase the probability of extinction. In general, disease and mortality patterns followed epidemic waves in the herds. However, an interesting pattern was seen for acute infections and abortions in adult cattle after the first 2 years of infection in herds with poor hygiene and high susceptibility. Repeated infections in young stock lead to a high proportion of resistant adult cattle, which lead to a dampening effect on acute infections in adults and associated abortions. Sensitivity analyses of 24 alternative scenarios showed that a super shedder state was not essential to mimic the infection dynamics and persistence patterns known from field studies, but a persistent carrier state was required in the model to mimic real life S. Dublin infections.

Long-term cortisol levels are not associated with nasal carriage of Staphylococcus aureus

Staphylococcus aureus (S. aureus) colonizes the anterior nares in part of the population and the persistent carrier state is associated with increased infection risk. Knowledge concerning the determinants of S. aureus nasal carriage is limited. Previously, we found that glucocorticoid receptor polymorphisms influence carrier risk, suggesting involvement of glucocorticoids. Our aim was to study long-term cortisol levels in non-carriers, intermittent, and persistent carriers of S. aureus. We hypothesized that cortisol levels are higher in carriers, since cortisol-induced immune suppression would enhance S. aureus colonization. We determined nasal carrier state and long-term hair cortisol levels in 72 healthy subjects. Nasal swabs were collected twice with an interval of 2 weeks. Cortisol levels were determined in hair segments of 3 cm, which corresponds to a period of roughly 3 months. Of all 72 participants, 38 were non-carriers, 10 were intermittent carriers, and 24 were persistent carriers of S. aureus. Cortisol levels did not differ between these carrier groups (p = 0.638). Long-term cortisol levels are not associated with S. aureus nasal carriage.

Lack of Correlation between Antibody Titers to Fibrinogen-Binding Protein of Streptococcus Equi and Persistent Carriers of Strangles

Previously published studies have neither used nor reported the results of an indirect enzyme-linked immunosorbent assay (iELISA) to measure serologic responses in natural outbreaks of strangles. The concept of using serologic responses to identify persistent carriers of Streptococcus equi has been proposed but not scientifically evaluated. The specific aims of the current study were to determine the duration and level of truncated fibrinogen-binding protein-specific (SeM allele 1) antibody production in ponies involved in a natural outbreak of strangles and to determine if test results from this serologic iELISA could predict persistent carrier status. Serologic samples were obtained before and after an outbreak of naturally occurring strangles infection. Persistent carriers of S. equi were identified via culture and polymerase chain reaction (PCR) testing of lavage fluid collected from the guttural pouches and nasopharynx or swabs of the nasopharynx after recovery from acute disease and at postmortem examination. Logistic regression analysis was used to determine if an association existed between serologic response and persistent carrier state. The ELISA reported in the current study definitively confirmed a recent exposure to S. equi. However, the measured serologic response did not predict carrier status in this strangles outbreak. Therefore, a guttural-pouch endoscopy with subsequent culture or PCR testing to detect S. equi remains the most accurate method available for the identification of persistent carriers.

Herd-level Management and Biosecurity Factors associated with Measures of Reproductive Success in California Beef Cow-calf Herds

A cross-sectional study was performed to evaluate the relationship between herd-level management and biosecurity practices in a sample of California's beef cow-calf operations, and the estimated within-herd prevalence of antibodies to Neospora caninum , Anaplasma marginale, and the persistent carrier state of bovine viral diarrhea virus (BVDV PI). Serum samples were collected from 917 adult beef cows from 29 herds. Multivariable linear and logistic regression models were created to predict calving season length and the probability of a cow weaning a calf using selected herd-level management and biosecurity factors and the seroprevalence data. The overall seroprevalence of A. marginale , N. caninum and BVDV PI was 47.4% (435/917), 8.9% (82/917) and 0.1% (1/917), respectively. The multiple linear regression analysis revealed no significant associations between management variables and calving season length. A logistic regression model found that the probability that a cow weaned a calf was associated with the number of first-calf heifers in the herd, whether the bulls were semen tested, whether the herd had a history of epizootic bovine abortion, and if the cow herd was vaccinated against Clostridium spp. Based on these findings, we conclude that herd-level management and biosecurity factors may be associated with reproductive success in beef cow-calf farms in California through their effect on the probability that a cow weans a calf.

Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection.

Primary virus infection often elicits a large CD8+ T cell response which subsequently contracts to a smaller memory T cell pool; the relationship between these two virus-specific populations is not well understood. Here we follow the human CD8+ T cell response to Epstein-Barr virus (EBV) from its primary phase in infectious mononucleosis (IM) through to the persistent carrier state. Using HLA-A2.1 or B8 tetramers specific for four lytic cycle and three latent cycle epitopes, we find marked differences in the epitope-specific composition of the T cell populations between the two phases of infection. The primary response is dominated by lytic epitope specificities which are severely culled (and in one case extinguished) with resolution of the acute infection; in contrast latent epitope specificities are less abundant, if present at all, in acute IM but often then increase their percentage representation in the CD8 pool. Even comparing epitopes of the same type, the relative size of responses seen in primary infection does not necessarily correlate with that seen in the longer term. We also follow the evolution of phenotypic change in these populations and show that, from a uniform CD45RA−RO+CCR7− phenotype in IM, lytic epitope responses show greater reversion to a CD45RA+RO− phenotype whereas latent epitope responses remain CD45RA−RO+ with a greater tendency to acquire CCR7. Interestingly these phenotypic distinctions reflect the source of the epitope as lytic or latent, and not the extent to which the response has been amplified in vivo.

Arenaviruses: protein structure and function.

The Arenaviruses are structurally quite simple, consisting of only four primary gene products encoded on two RNA strands. Despite this very simple genetic plan, these viruses are capable of interacting with the host in a bewildering variety of permutations that result in a range of infections from a lifelong persistent carrier state, as is evident in the neonatally or congenitally infected carrier mouse, to the lethal acute diseases typical of Lassa, Machupo, Junin, and other arenavirus hemorrhagic fevers. These differing disease courses are, in part, determined by the clash of two factors: the cellular and tissue site(s) of virus replication in vivo, defined as tropism, and the genetically determined host response to the infecting virus. When the virus replicates in critical tissues like the meninges and choroid plexi of the mouse brain, as is the case in acute LCMV infection, and the host is capable of mounting a strong T-cell response, the end result is a fatal choriomeningitis (Fig. 1). However, replication of the virus in these same tissues results in little tissue injury or disease in a mouse that has been immunosuppressed, or in a neonate prior to maturation of the T-cell response. This immunopathological paradox is at the core of understanding the pathogenesis of LCM disease and has provided a singularly important biological model for probing cellular immunology for more than six decades (Buchmeier and Zajac 1999). In this brief chapter, the nature of the arenavirus proteins and their functions will be discussed with particular emphasis on their role in infection and disease.

Toward prevention of hepatocellular carcinoma developing in chronic hepatitis C

Toward prevention of hepatocellular carcinoma developing in chronic hepatitis C

Theileria annulata: carrier state and immunity.

Recovery from primary infection of Theileria annulata results in the development of a persistent carrier state in the vertebrate host. The carrier state is of great importance in the maintenance of the life cycle by alternate tick/cattle challenge and both contributes to and may be necessary for maintenance of immunity. Therefore, an accurate determination of carrier animals could be useful in determining immune status and may allow the necessary control measures to be implemented. Detailed information on the carrier state of animals following immunization with attenuated cell lines is lacking. In this study, relationship between immune response, persistence of the parasite, and the antibody response has been investigated. Calves were infected with T. annulata sporozoites, low passage (non-attenuated) or high passage (attenuated, vaccine) cell lines and later challenged with a lethal dose of heterologous sporozoites. The presence and persistence of the parasite were monitored by PCR using primers derived from genes coding for ssrRNA and a 30 kDa major merozoite surface protein, by Giemsa stained blood smears to detect the presence of piroplasms and also by attempting to establish infected mononuclear cell cultures from venous blood. Antibody responses were measured by indirect ELISA using a merozoite recombinant antigen and IFAT using piroplasm and macroschizont antigens. Results showed that there was an evident relationship between the persistence of carrier status, antibody response in ELISA and immune response to challenge.

Loss of circulating hepatitis C virus in children who developed a persistent carrier state after mother-to-baby transmission.

Of the 15 babies born to mothers infected with hepatitis C virus (HCV) and followed since birth, three developed HCV RNA in their serum. HCV RNA disappeared in two infants within 2 mo, but it persisted in the remaining infant. Mother-to-baby transmission was diagnosed retrospectively in an additional eight children aged 0.8-13.6 y. The eight children were followed for 1.4-5.0 y (mean +/- SD: 3.2 +/- 1.3 y) until they were 3.3-16.7 y old (8.5 +/- 4.3 y). Serum HCV RNA disappeared and antibodies to HCV decreased in the titer in two of the children when they were 3 y old. The spontaneous loss of serum HCV RNA was not observed in any of the other 14 children with posttransfusion infection who were followed for 2.6-6.1 y (4.0 +/- 1.1 y), until 3-22 y from the time they received transfusions and when they were 8.4-22.8 y old (15.4 +/- 4.1 y). These results indicate that the vertical transmission of HCV is rare, and some children can resolve the infection after a few years, whereas the infection persists in children who are infected by transfusion.

Alteration of neutrophil function in BCG-treated and non-treated swine after exposure to Salmonella typhimurium

Salmonella typhimurium infection in swine causes an enterocolitis followed by a persistent carrier state, but little is known about the mechanisms that allow this organism to colonize and persist in host tissues. Neutrophils provide a first line of defense against invading pathogens such as Salmonella typhimurium. The purpose of this study was to evaluate porcine neutrophil function after in vivo exposure to Salmonella and to determine if the immunomodulator, bacillus Calmette Guerin (BCG), exerts any effect on neutrophil function or on the colonization and persistence of S. typhimurium in the pig. Compared to negative controls, neutrophils from pigs exposed to S. typhimurium exhibited significantly decreased iodination, cytochrome-C reduction, antibody-dependent cell-mediated cytotoxicity, random migration, and chemotaxis ( P≤0.05). Neutrophil bactericidal activity against S. typhimurium was significantly enhanced. Most of the significant differences were noted in the first two days after exposure to Salmonella. Often the functional alterations were biphasic, peaking again 7–10 days after exposure. BCG alone significantly depressed random migration and cytochrome-C reduction in unstimulated neutrophils. The clinical course, colonization pattern, and persistence of Salmonella were similar between pigs receiving BCG and untreated pigs. These data suggest that S. typhimurium infection causes a depression in oxidative metabolism and motility, yet an increase in overall bactericidal activity against S. typhimurium in circulating porcine neutrophils. It also appears that BCG treatment, as reported here, does not enhance resistance of pigs to S. typhimurium colonization or reduce the number of persistent organisms in the porcine ileum.

Frequency and significance of concurrent hepatitis B surface antigen and antibody in acute and chronic hepatitis B

Of 228 consecutive hepatitis B surface antigen (HBsAg)-positive patients who had simultaneous hepatitis B surface antibody (anti-HBs) determinations, HBsAg and anti-HBs were found concurrently in 73 (32%). Concurrence was found with greater frequency in patients with chronic active hepatitis than in those with acute hepatitis (36 57 vs. 13 38, p < 0.02). Patients with chronic active hepatitis had concurrent markers more commonly than those with chronic persistent hepatitis or asymptomatic carrier state (36 57 vs. 24 133, p < 0.001). No major risk factors were identified. Hepatitis B e antigen was detected more frequently in patients with concurrence (68% vs. 42%, p < 0.01). Subtyping was possible in 30 patients with chronic infection, including 18 with chronic active hepatitis, 7 with chronic persistent hepatitis, and 5 with a carrier state. In 25 patients, HBsAg subtype ad was found with antibody to subdeterminant y and in four instances, HBsAg subtype ay was found with antibody to subdeterminant d. Only 1 patient had homotypic HBsAg and anti-HBs. Of 38 patients who had successive determinations, concurrence was constant in 29. In 9 others, anti-HBs was detected intermittently and the heterotypia of the recurrent antibody remained constant. Antibody to hepatitis δ-virus was not detected. We conclude that concurrent HBsAg and anti-HBs are found frequently in acute and chronic hepatitis B. The markers are commonly heterotypic in chronic disease. The presence of heterotypic markers is not associated with specific risk factors or δ-infection. Concurrence is associated with evidence of viral replication and features of active inflammation.

PREVENTION OF THE HBsAg CARRIER STATE IN NEWBORN INFANTS OF MOTHERS WHO ARE CHRONIC CARRIERS OF HBsAg AND HBeAg BY ADMINISTRATION OF HEPATITIS-B VACCINE AND HEPATITIS-B IMMUNOGLOBULIN: Double-blind Randomised Placebo-controlled study

Newborn infants of Chinese HBeAg-carrier mothers in Hong Kong were randomly assigned to one of four study groups. Group I was treated with 3 μg heat-inactivated hepatitis B (HB) vaccine at birth and at 1, 2, and 6 months thereafter, in conjunction with seven monthly HBIg injections; group II was treated according to the same vaccine schedule but received only one HBIg injection at birth; group III received only the vaccine, at months 0, 1, 2, and 6; and group IV received placebos for both vaccine and HBIg. The first set of injections was given within 1 h after birth. Comparisons were made in the 140 children who were at least six months old at the close of the trial (495 days). In all three treatment groups development of the persistent carrier state was significantly (p0·0001) less frequent than in controls (2·9%, 6·8%, and 21·0% versus 73·2%). Although vaccination alone was significantly less protective than vaccination plus multiple HBIg injections (p=0·03), the degree of protection was still remarkable. 12 months after the first set of injections 96-100% of the infants in the three treatment groups were anti-HBs positive; the geometric mean titres of anti-HBs in the three groups did not differ significantly. This indicates that even high doses of HBIg do not interfere with the anti-HBs response to the vaccine. Probable intra-uterine HB infections were observed in 3 infants. No serious side-effects were observed from the interventions, even in the babies with intra-uterine infections who had received HBIg and HB-vaccine at birth. To prevent development of the persistent HBsAg carrier state, and thereby the consequent chronic liver disease and/or primary carcinoma of the liver, HB vaccine and HBIg should be administered as soon as possible after birth to all newborn infants at risk of perinatal hepatitis B infection.

Isolation of a heterogeneous population of temperature-sensitive mutants of measles virus from persistently infected human lymphoblastoid cell lines.

Two human lymphoblastoid B-cell lines, WI-L2 and 8866, were infected with the Edmonston strain of measles virus at a multiplicity of infection of 10(-6), and stable persistent infections were established. By immunofluorescence and electron microscopy, the vast majority of cells from both cell lines were expressing viral antigens and releasing virion-like particles. However, very little infectious virus could be detected at 37 degrees C, either by an infectious centers assay or by titration of supernates from persistently infected cultures. When cultures were shifted to 31 degrees C, the cells released a population of virus that was temperature-sensitive. Clonal analysis of supernatant virus at 31 degrees C revealed a highly heterogeneous population of temperature-sensitive mutants, differing in plating efficiency ratios, thermolability, and antigen production at the nonpermissive temperature. Factors such as interferon, defective interfering particles, and extracellular virus do not appear to be important in maintaining the persistent carrier state. These studies have important implications for persistent infections of lymphoid cells in vivo, and the slow neurological diseases associated with measles, subacute sclerosing panencephalitis, and multiple sclerosis.