Mathew D. Esona

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

BACKGROUNDnWe 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.nnnMETHODSnWe 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.nnnRESULTSnRV5-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].nnnCONCLUSIONSnBoth RV5 and RV1 significantly protected against medically attended rotavirus gastroenteritis in this real-world assessment.

Long-term Consistency in Rotavirus Vaccine Protection: RV5 and RV1 Vaccine Effectiveness in US Children, 2012–2013

BACKGROUNDnUsing a multicenter, active surveillance network from 2 rotavirus seasons (2012 and 2013), we assessed the vaccine effectiveness of RV5 (RotaTeq) and RV1 (Rotarix) rotavirus vaccines in preventing rotavirus gastroenteritis hospitalizations and emergency department (ED) visits for numerous demographic and secular strata.nnnMETHODSnWe enrolled children hospitalized or visiting the ED with acute gastroenteritis (AGE) for the 2012 and 2013 seasons at 7 medical institutions. Stool specimens were tested for rotavirus by enzyme immunoassay and genotyped, and rotavirus vaccination histories were compared for rotavirus-positive cases and rotavirus-negative AGE controls. We calculated the vaccine effectiveness (VE) for preventing rotavirus associated hospitalizations and ED visits for each vaccine, stratified by vaccine dose, season, clinical setting, age, predominant genotype, and ethnicity.nnnRESULTSnRV5-specific VE analyses included 2961 subjects, 402 rotavirus cases (14%) and 2559 rotavirus-negative AGE controls. RV1-specific VE analyses included 904 subjects, 100 rotavirus cases (11%), and 804 rotavirus-negative AGE controls. Over the 2 rotavirus seasons, the VE for a complete 3-dose vaccination with RV5 was 80% (confidence interval [CI], 74%-84%), and VE for a complete 2-dose vaccination with RV1 was 80% (CI, 68%-88%).Statistically significant VE was observed for each year of life for which sufficient data allowed analysis (7 years for RV5 and 3 years for RV1). Both vaccines provided statistically significant genotype-specific protection against predominant circulating rotavirus strains.nnnCONCLUSIONSnIn this large, geographically and demographically diverse sample of US children, we observed that RV5 and RV1 rotavirus vaccines each provided a lasting and broadly heterologous protection against rotavirus gastroenteritis.

One Year Survey of Human Rotavirus Strains Suggests the Emergence of Genotype G12 in Cameroon

In this study the emergence of rotavirus A genotype G12 in children <5 years of age is reported from Cameroon during 2010/2011. A total of 135 human stool samples were P and G genotyped by reverse transcriptase PCR. Six different rotavirus VP7 genotypes were detected, including G1, G2, G3, G8, G9, and G12 in combinations with P[4], P[6] and P[8] VP4 genotypes. Genotype G12 predominated in combination with P[8] (54.1%) and P[6] (10.4%) genotypes followed by G1P[6] (8.2%), G3P[6] (6.7%), G2P[4] (5.9%), G8P[6] (3.7%), G2P[6] (0.7%), G3P[8] (0.7%), and G9P[8] (0.7%). Genotype P[6] strains in combination with various G‐types represented a substantial proportion (Nu2009=u200944, 32.6%) of the genotyped strains. Partially typed strains included G12P[NT] (2.2%); G3P[NT] (0.7%); G(NT)P[6] (1.5%); and G(NT)P[8] (0.7%). Mixed infections were found in five specimens (3.7%) in several combinations including G1u2009+u2009G12P[6], G2u2009+u2009G3P[6]u2009+u2009P[8], G3u2009+u2009G8P[6], G3u2009+u2009G12P[6]u2009+u2009P[8], and G12P[6]u2009+u2009P[8]. The approximately 10% relative frequency of G12P[6] strains detected in this study suggests that this strain is emerging in Cameroon and should be monitored carefully as rotavirus vaccine is implemented in this country, as it shares neither G‐ nor P‐type specificity with strains in the RotaTeq® and Rotarix® vaccines. These findings are consistent with other recent reports of the global spread and increasing epidemiologic importance of G12 and P[6] strains. J. Med. Virol. 85:1485–1490, 2013.

Full genome characterization of human Rotavirus A strains isolated in Cameroon, 2010-2011: diverse combinations of the G and P genes and lack of reassortment of the backbone genes.

Over the past few years whole genome sequencing of rotaviruses has become a routine laboratory method in many strain surveillance studies. To study the molecular evolutionary pattern of representative Cameroonian Rotavirus A (RVA) strains, the semiconductor sequencing approach was used following random amplification of genomic RNA. In total, 31 RVA strains collected during 2010-2011 in three Cameroonian study sites located 120 to 1240 km from each other were sequenced and analyzed. Sequence analysis of the randomly selected representative strains showed that 18 RVAs were Wa-like, expressing G1P[6], G12P[6], or G12P[8] neutralization antigens on the genotype 1 genomic constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1), whereas 13 other strains were DS-1-like, expressing G2P[4], G2P[6], G3P[6], and G6P[6] on the genotype 2 genomic constellation (I2-R2-C2-M2-A2-N2-T2-E2-H2). No inter-genogroup reassortment in the backbone genes was observed. Phylogenetic analysis of the Cameroonian G6P[6] strains indicated the separation of the strains identified in the Far North region (Maroua) and the Northwest region (Bamenda and Esu) into two branches that is consistent with multiple introductions of G6P[6] strains into this country. The present whole genome based molecular characterization study indicates that the emerging G6P[6] strain is fully heterotypic to Rotarix, the vaccine introduced during 2014 in childhood immunization program in Cameroon. Continuous strain monitoring is therefore needed in this area and elsewhere to see if G6s, besides genotype G1 to G4, G8, G9 and G12, may become a new, regionally important genotype in the post vaccine licensure era in Africa.

Comparative evaluation of commercially available manual and automated nucleic acid extraction methods for rotavirus RNA detection in stools

n Abstractn n Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS® easyMAG® instruments, the NucliSENS® miniMAG® semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid® kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols.n n

Virus Detection and Duration of Illness Among Patients With 2009 Pandemic Influenza A (H1N1) Virus Infection in Texas

Knowledge from early outbreaks is limited regarding the virus detection and illness duration of the 2009 pandemic influenza A (H1N1) infections. During the period from April to May 2009 in Texas, we collected serial nasopharyngeal (NP) and stool specimens from 35 participants, testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and culture. The participants were aged 2 months to 71 years; 25 (71%) were under 18. The median duration of measured fever was 3.0 days and of virus detection in NP specimens was 4.2 days; however, few specimens were collected between days 5-9. The duration of virus detection (4.2 days) was similar to the duration of fever (3.5 days) (RR, 1.14; 95% CI, .66-1.95; P =u2009.8), but was shorter than the duration of cough (11.0 days) (RR, .41; 95% CI, .24-.68; P <u2009.001). We detected viral RNA in two participants stools. All cultures were negative. This investigation suggests that the duration of virus detection was likely similar to the seasonal influenza virus.

Molecular surveillance of rotavirus strains circulating in Yaoundé, Cameroon, September 2007-December 2012.

Rotavirus is the most common cause of severe diarrheal disease in children under 5 years of age worldwide. The World Health Organization (WHO) estimated that 453,000 rotavirus-attributable deaths occur annually. Through the WHO, the Rotavirus Sentinel Surveillance Program was established in Cameroon in September 2007 with the Mother and Child Center (MCC) in Yaoundé playing the role of sentinel site and national laboratory for this program. The objectives of this surveillance were to assess the rotavirus disease burden and collect baseline information on rotavirus strains circulating in Cameroon. Diarrheal stool samples were collected in a pediatric hospital from children under 5, using the WHO case definition for rotavirus diarrhea. Antigen detection of rotavirus was performed by using an enzyme immunoassay (EIA). The genotypic characterization was performed using multiplexed semi-nested reverse transcription-polymerase chain reaction (RT-PCR) assays. Between September 2007 and December 2012, 2444 stool samples were received at the MCC laboratory for rotavirus antigen detection, of which 999 (41%) were EIA positive. Among EIA positive samples 898 were genotyped. Genotype prevalence varied each year. Genotype G9P[8] was the dominant type during 2007 (32%) and 2008 (24%), genotype G3P[6] predominated in 2010 (36%) and 2011 (25%), and G1P[8] was predominant in 2012 (44%). The findings showed that the rotavirus disease burden is high and there is a broad range of rotavirus strains circulating in Yaoundé. These data will help measure the impact of vaccination in the future.

Detection of novel porcine bocaviruses in fecal samples of asymptomatic pigs in Cameroon

Improvements and widespread use of nucleic acid amplification and sequencing methods have led to the recognition of new virus diversity in various domestic animals, including pigs. In this study we utilized either virus species specific or broadly reactive PCR assays to describe the occurrence and genetic diversity of selected DNA viruses belonging to families Adenoviridae, Circoviridae, Anelloviridae and Parvoviridae in Cameroonian pigs. Fecal specimens were collected during spring of 2011. No adenoviruses, circoviruses and anelloviruses were detected, however, high prevalence and remarkable genetic diversity within the identified parvoviruses and, particularly, within bocaviruses was observed. PPV4 was the most prevalent virus (20%), followed by PBoV3 (18%), PBoV4 (18%), PBoV5 plus 6V/7V (16%), and PBoV1 plus PBoV2 (6%). The frequency of mixed infections with various combinations of these virus species reached 20%. Genetic analysis of the identified viruses showed that the capsid gene of PBoV1 and PBoV2 strains shared up to 91% and 94%nt sequence similarities to reference PBoV1 and PBoV2 strains, respectively. The identified PBoV3 and PBoV4 strains shared ≤ 95% and ≤ 98%nt identities with reference PBoV3 and PBoV4 strains, respectively, along the NS gene, whereas the PBoV5 strains shared 86%nt identities with Hungarian and 87%nt identities with Chinese PBoV5 strains along the capsid gene. In addition, a single PBoV5-like strain shared ≤ 71%nt sequence identity with other PBoV5 strains. This is the first study to report evidence of the circulation of bocaviruses in Africa and contributes to our understanding of the impact of globalization on the dispersal of new and emerging viruses.

Expression and characterization of human group C rotavirus virus-like particles in insect cells☆

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.

Detection of PCV-2 DNA in stool samples from infants vaccinated with RotaTeq®.

Rotarix® and RotaTeq® vaccines have led to a dramatic reduction in rotavirus disease worldwide. However, the detection of porcine circovirus type 1 (PCV-1) and 2 (PCV-2) DNA in these vaccines raised some safety concerns. Studies examining shedding of rotavirus in stool from rotavirus vaccine recipients have been performed but no published data exist regarding the shedding of PCV virus in stools of vaccinees. The goal of this study was to determine if PCV-1 and/or PCV-2 is shed in the feces of infants vaccinated with RotaTeq®. Using multiple PCR assays for detection of PCV DNA, we tested for PCV-1 and PCV-2 in 826 stool swab samples collected serially during the first 9 d after vaccination from 102 children vaccinated with RotaTeq®. Since the vaccine is recommended and uptake is high, we did not have samples from unvaccinated infants. A total of 235 (28.5%) samples from 59 vaccine recipients were positive for PCV-2 DNA by one or more assays used in this study. PCV-1 DNA was not detected in RotaTeq® or any of the stool swab extracts. Twenty-two of the 102 vaccine recipients (21.6%) shed RotaTeq® vaccine strain and 10 of these vaccinees (9.8%) were shedding both PCV DNA and rotavirus vaccine RNA. PCV DNA was detected up to 9 d post vaccination and was most frequently detected in the first 5 d after vaccination. This study demonstrated shedding of PCV-2 DNA by RotaTeq® vaccinees but we did not find evidence that this DNA was associated with viable PCV. Findings from this study support the continued use of current rotavirus vaccines.