Michael D. Bowen

Genetic Diversity among Lassa Virus Strains

ABSTRACT The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a “molecular clock” was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.

Effectiveness of rotavirus vaccination against childhood diarrhoea in El Salvador: case-control study

Objective To evaluate the effectiveness of a monovalent rotavirus vaccine against severe rotavirus disease and to assess its impact on diarrhoea in children aged less than 2 years after national introduction in El Salvador, a low-middle income country in Central America. Design Matched case-control study. Setting Seven hospitals in cities across El Salvador, January 2007 to June 2009. Participants 323 children aged less than 2 years admitted with laboratory confirmed rotavirus diarrhoea and 969 healthy controls matched for age and neighbourhood. Main outcome measure Effectiveness of rotavirus vaccination ((1–adjusted odds ratio of vaccination)×100) against rotavirus diarrhoea requiring hospital admission. Results Cases and controls were similar for breast feeding, premature birth, maternal education, and socioeconomic variables. G1P[8] strains were identified in 92% of rotavirus cases. Effectiveness of two doses of vaccination against diarrhoea requiring hospital admission was 76% (95% confidence interval 64% to 84%). Protection was significantly lower (P=0.046) among children aged 12 months or more (59%, 27% to 77%) compared with children aged 6-11 months (83%, 68% to 91%). One dose of vaccine was 51% (26% to 67%) effective. At the sentinel hospitals, all admissions for diarrhoea among children under 5 declined by 40% in 2008 and by 51% in 2009 from the prevaccine year 2006. Conclusions A monovalent rotavirus vaccine was highly effective against admissions for rotavirus diarrhoea in children aged less than 2 years in El Salvador and substantially reduced the number of such admissions in this low-middle income setting. The impact on disease epidemiology after vaccination, particularly among older children, warrants future attention.

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.

United States rotavirus strain surveillance from 2005 to 2008: genotype prevalence before and after vaccine introduction.

Background: A live, attenuated rotavirus vaccine, RotaTeq®, was approved in 2006 for immunization of infants in the United States. To monitor the distribution of rotavirus genotypes before and after vaccine introduction, the Centers for Disease Control and Prevention conducted strain surveillance with the National Rotavirus Strain Surveillance System. Methods: Over 3 rotavirus seasons, 2005–2006, 2006–2007, and 2007–2008, National Rotavirus Strain Surveillance System laboratories collected rotavirus-positive stool specimens and submitted them to the Centers for Disease Control and Prevention. Rotavirus strains were G- and P-genotyped by multiplex reverse transcription-polymerase chain reaction or nucleotide sequencing. Results: During 2005–2006 and 2006–2007 seasons, G1 was the dominant G-type but in the 2007–2008 season, G3 replaced G1 as the most frequently detected strain. Four genotypes, G1P[8], G2P[4], G3P[8], and G9P[8] were detected in every season. Uncommon strains observed during the study period were G2P[8], G1P[6], G2P[6], G4P[6], G1P[4], G3P[9], G12P [6], and G12P[8]. The mean age of rotavirus cases in the 2007–2008 season increased significantly in patients less than 3 years old compared with the 2 previous seasons. Conclusions: The increased overall prevalence of G3P [8] strains in 2007–2008, the first rotavirus season with reasonable rotavirus vaccine coverage, was consistent with Australian reports of G3 dominance following RotaTeq introduction. However, these strain changes in both countries have occurred in the context of large declines in severe rotavirus disease and we cannot rule out that they are simply the result of naturally occurring changes in rotavirus strain prevalence. These findings underscore the need for careful monitoring of strains to assess possible vaccine pressure-induced changes and vaccine effectiveness against various rotavirus genotypes.

Effectiveness of Pentavalent and Monovalent Rotavirus Vaccines in Concurrent Use Among US Children <5 Years of Age, 2009–2011

BACKGROUND We assessed vaccine effectiveness (VE) for RotaTeq (RV5; 3 doses) and Rotarix (RV1; 2 doses) at reducing rotavirus acute gastroenteritis (AGE) inpatient and emergency department (ED) visits in US children. METHODS We enrolled children <5 years of age hospitalized or visiting the ED with AGE symptoms from November 2009-June 2010 and from November 2010-June 2011 at 7 medical institutions. Fecal specimens were tested for rotavirus by enzyme immunoassay and genotyped. Vaccination among laboratory-confirmed rotavirus cases was compared with rotavirus-negative AGE controls. Regression models calculated VE estimates for each vaccine, age, ethnicity, genotype, and clinical setting. RESULTS RV5-specific analyses included 359 rotavirus cases and 1811 rotavirus-negative AGE controls. RV1-specific analyses included 60 rotavirus cases and 155 rotavirus-negative AGE controls. RV5 and RV1 were 84% (95% confidence interval [CI], 78%-88%) and 70% (95% CI, 39%-86%) effective, respectively, against rotavirus-associated ED visits and hospitalizations combined. By clinical setting, RV5 VE against ED and inpatient rotavirus-associated visits was 81% (95% CI, 70%-84%) and 86% (95% CI, 74%-91%), respectively. RV1 was 78% (95% CI, 46%-91%) effective against ED rotavirus disease; study power was insufficient to evaluate inpatient RV1 VE. No waning of immunity was evident during the first 4 years of life for RV5, nor during the first 2 years of life for RV1. RV5 provided genotype-specific protection against each of the predominant strains (G1P[8], G2P[4], G3P[8], G12P[8]), while RV1 VE was statistically significant for the most common genotype, G3P[8]. CONCLUSIONS Both RV5 and RV1 significantly protected against medically attended rotavirus gastroenteritis in this real-world assessment.

Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

Retrospective serological and genetic study of the distribution of hantaviruses in Greece

A retrospective serological and genetic study of hantaviruses responsible for hemorrhagic fever with renal syndrome (HFRS) in Greece during the last 17 years is presented. Fifty‐one serum samples taken from 30 HFRS cases previously diagnosed by immunofluorescence assay were tested by ELISA for IgG (Hantaan, Dobrava, and Puumala) and IgM antibodies (Hantaan and Puumala). Results were compatible with the majority of infections being related to hantaviruses carried by rodents of the subfamily Murinae. RNA was extracted from 26 selected samples and reverse transcriptase‐polymerase chain reaction (RT‐PCR) was performed using primers specifically designed for the detection of hantaviruses associated with murine (MS‐N‐specific, MM‐G1‐specific primers) or arvicoline rodents (PPT‐N‐specific primers). In addition, primers previously designed for the detection of the G2 coding region of the Murinae‐associated hantaviruses were also used. Sequencing of the PCR products was then performed, followed by phylogenetic analysis of nucleotide sequence differences. Eleven out of the 26 serum samples tested were found to be positive by PCR with the MS‐N primers, whereas four were positive with the MM‐G1 primers, and only two with the G2 primers. None of the samples was found positive with the PPT primers. The sequence analysis showed that the virus that was responsible for these 11 HFRS cases was the Dobrava virus, which is endemic throughout the Balkans. J. Med. Virol. 55:321–327, 1998.

Sibling Transmission of Vaccine-Derived Rotavirus (RotaTeq) Associated With Rotavirus Gastroenteritis

Although rotavirus vaccines are known to be shed in stools, transmission of vaccine-derived virus to unvaccinated contacts resulting in symptomatic rotavirus gastroenteritis has not been reported to our knowledge. We document here the occurrence of vaccine-derived rotavirus (RotaTeq [Merck and Co, Whitehouse Station, NJ]) transmission from a vaccinated infant to an older, unvaccinated sibling, resulting in symptomatic rotavirus gastroenteritis that required emergency department care. Results of our investigation suggest that reassortment between vaccine component strains of genotypes P7[5]G1 and P1A[8]G6 occurred during replication either in the vaccinated infant or in the older sibling, raising the possibility that this reassortment may have increased the virulence of the vaccine-derived virus. Both children remain healthy 11 months after this event and are without underlying medical conditions.

Effectiveness of Monovalent and Pentavalent Rotavirus Vaccine

OBJECTIVE: Previous US evaluations have not assessed monovalent rotavirus vaccine (RV1, a G1P[8] human rotavirus strain) effectiveness, because of its later introduction (2008). Using case-control methodology, we measured the vaccine effectiveness (VE) of the 2-dose RV1 and 3-dose pentavalent vaccine (RV5) series against rotavirus disease resulting in hospital emergency department or inpatient care. METHODS: Children were eligible for enrollment if they presented to 1 of 5 hospitals (3 in Georgia, 2 in Connecticut) with diarrhea of ≤10 days’ duration during January through June 2010 or 2011, and were born after RV1 introduction. Stools were collected; immunization records were obtained from providers and state electronic immunization information system (IIS). Case-subjects (children testing rotavirus antigen-positive) were compared with 2 control groups: children testing rotavirus negative and children selected from IIS. RESULTS: Overall, 165 rotavirus-case subjects and 428 rotavirus-negative controls were enrolled. Using the rotavirus-negative controls, RV1 VE was 91% (95% confidence interval [CI] 80 to 95) and RV5 VE was 92% (CI 75 to 97) among children aged ≥8 months. The RV1 VE against G2P[4] disease was high (94%, CI 78 to 98), as was that against G1P[8] disease (89%, CI 70 to 96). RV1 effectiveness was sustained among children aged 12 through 23 months (VE 91%; CI 75 to 96). VE point estimates using IIS controls were similar to those using rotavirus-negative controls. CONCLUSIONS: RV1 and RV5 were both highly effective against severe rotavirus disease. RV1 conferred sustained protection during the first 2 years of life and demonstrated high effectiveness against G2P[4] (heterotypic) disease.

Reassortant Group A Rotavirus from Straw-colored Fruit Bat (Eidolon helvum)

TOC summary: Bats may be reservoirs of zoonotic viruses that threaten human health.