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Pandemic vaccine seed viruses for H5N1, H5N2 and H9N2 subtypes were prepared and evaluated. Formalin-inactivated reassortant vaccines using A/Puerto Rico/8/34 (PR8) as a donor of internal genes with surface glycoprotein genes, hemagglutinin (HA) and neuraminidase (NA), of H5N2 and H9N2 subtypes, were generated by conventional reassortment. A reassortant virus for the highly pathogenic H5N1 subtype was prepared using reverse genetics with a genetically modified HA gene. All candidate vaccines were immunogenic in mice and ferrets, and mice immunized with inactivated vaccines were completely protected against challenge infection with wild-type parent viruses. All viruses were non-pathogenic for chickens in the standard USDA pathotyping test.

We estimated annual numbers and rates of influenza-associated deaths and hospitalizations by age group in the United States using a Poisson regression model incorporating World Health Organization (WHO) viral surveillance data. Mortality data were obtained between 1976 and 2000 from the National Center for Health Statistics (NCHS); hospitalization data were obtained between 1979 and 2001 from the National Hospital Discharge Survey (NHDS). Weekly influenza isolates by type and subtype were obtained from the WHO collaborating laboratories for the 1976–1977 through 1999–2000 seasons. Our models estimated annual averages of 6796 underlying pneumonia and influenza deaths and 28,076 underlying respiratory and circulatory deaths. The models estimated annual averages of 88,479 primary pneumonia and influenza hospitalizations and 225,985 primary respiratory and circulatory hospitalizations. We found significant increases in the numbers of influenza-associated deaths and hospitalizations by year among persons aged ≥65 years due to the aging of the population, the predominance of A(H3N2) viruses during the late 1990s and the increasing length of the influenza season during the 1990s.

In 1997, we were ill prepared to produce vaccines in response to the sudden emergence of highly pathogenic H5N1 influenza. This experience stimulated efforts to improve technical and regulatory procedures for pandemic vaccine virus development and, in February 2003, when highly pathogenic H5N1 influenza re-appeared in man, we were able to produce H5N1 vaccine strains by reverse genetics within the space of 3 months. The technical and regulatory progress which made this possible is reviewed and newly developed WHO biosafety risk assessment for vaccine production is described. Recommendations are made for the significant challenges that lie ahead.

To prepare candidate influenza pandemic vaccines, we are developing an approach based on reassortment of antigenically appropriate nonpathogenic avian viruses of different subtypes (H5, H9, H7) with the cold-adapted master strain (MS) A/Leningrad/134/17/57 (Len/17) that is currently used in Russia for preparing licensed live attenuated vaccines for adults and children. In the present study, reassortants between A/Duck/Potsdam/1402-6/86(H5N2) (H5N2-wt) and Len/17 were obtained. One of the clones, A/17/Duck/Potsdam/86-92(H5N2) (Len17/H5), was chosen for further detailed genetic and antigenic analysis. Len17/H5 inherited the HA gene from the H5N2-wt and all other genes from Len/17 (7:1 genome composition). The HA gene sequence of Len17/H5 was identical to that of the parent H5N2-wt virus. The antigenic profile of the reassortant virus was similar to that of the H5N2-wt parent strain in the hemagglutination-inhibition (HI) test with a panel of antisera to different avian and human H5 viruses. The reassortant demonstrated high growth ability (9.3+0.3 lg EID50/ml) in embryonated hens' eggs (CE) at optimal (34 °C) temperature, comparable with that of the parent Len/17 MS. Also, Len17/H5 demonstrated cold-adapted (ca) and temperature-sensitive (ts) phenotypes similar to those of Len/17 and was attenuated for mice.

Pandemic influenza H2N2 viruses emerged in humans in 1957 and caused widespread morbidity and mortality in humans until 1968 when they were displaced by emerging H3N2 viruses. Although it is known that both the appearance and disappearance of H2N2 viruses involved reassortment between human and avian influenza viruses, genetic characterization of these viruses is limited. In this study, detailed genetic analysis of all eight gene segments of human H2N2 viruses isolated from 1957 until 1968 from geographically diverse regions was undertaken to establish a better understanding of the evolutionary nature of this virus. In addition, a number of human H3N2 viruses isolated from 1968 until 1972 were examined to investigate genetic events associated with the emergence of pandemic H3N2 viruses in humans. Phylogenic analysis of all gene segments of human H2N2 viruses consistently demonstrated divergent evolution. Genes of late H2N2 isolates were located in either of two distinct clades (I and II). Analysis of H3N2 viruses of 1968 revealed that all gene segments that were retained from H2N2 viruses were most similar to H2N2 virus genes of clade I. However, genes of both lineages were found to cocirculate among H3N2 isolates of 1969–1971. Furthermore, each gene segment demonstrated unique phylogenic topologies, indicating multiple reassortment events between late H2N2 and/or H3N2 viruses. The H3N2 viruses of 1972 analyzed here appeared to possess the genome constellation that represents the ancestral virus of contemporary H3N2 viruses. This constellation was first observed among isolates of 1970 and was distinct from that found among the earliest human H3N2 viruses from 1968. This evidence demonstrates that establishment of H3N2 viruses in humans was associated with multiple-reassortment events that contributed to genetic diversity among viruses.

Molecular studies of influenza virus isolated during 1996–2000, from 0- to 85-year-old patients with influenza-like illness living in the southern, southeastern, central western, northern and northeastern regions of Brazil, are described. Molecular epidemiology of the influenza virus demonstrated that strains H1N1 (A/Texas/36/91, A/Bayern/07/95, A/Taiwan/01/86, A/New Caledonia/20/99 and A/Johannesburg/82/99), detected from 1996 through 2000, evolved gradually from the strain A/Taiwan/1/86. Similarly, the strain H3N2 (A/Alaska/10/95, A/Johannesburg/33/94, A/Wuhan/35/95, A/Sydney/05/97, A/Moscow/10/99 and A/Panama/2007/99) seems to have evolved gradually from the ancestral strain A/Beijing/32/92. With regard to influenza virus type B, the strains B/Beijing/184/93 and B/Sichuan/279/99, evolved gradually from the ancestral B/Yamagata/26/88. Thus, molecular studies have showed that influenza viruses circulating from 1996 to 2000 in Brazil were similar to those on other continents. In addition, this investigation emphasizes the fundamental role of surveillance in order to select appropriate influenza strains for Southern Hemisphere vaccine composition.

Background: This study investigates the changes in human flu viruses in areas where unique animal flu viruses or epidemics were isolated in Taiwan and Kinmen islet. Materials and methods: Sentinel physicians obtained throat swabs from patients presenting with flu-like illness and two-step RT-PCR, using five sets of primers for matrix (M) and HA2, to detect all the animal influenza viruses and then typing/subtyping for human flu viruses. Questionnaires included travel history, animal contacts and occupations. Results: One flu B and eight flu A H3N2 isolates were identified in the flu season of 2002–2003, including 1, 2, 2 and 3 H3N2 in Taoyuan, Yilan, Tainan and Kinmen, respectively. Amino acid sequences of HA of human isolates revealed that Pro (P) at position 227 among most of the Kinmen isolates (2/3) replaced Ser (S) in the Taiwanese isolates, including one mutant from Gln (Q) to His (H) at position 156 of the HA gene for five out of eight H3N2 Taiwan isolates. Phylogenetic analysis demonstrated that all of the H3N2 viruses were of human origin (belonging to the lineage of A/Fujian/411/2002), with 80–90% homology in HA, NA and M gene segments of A/Swine/PingTung/Taiwan/199.2/02 (H3N2) and 75–77% homology in HA gene segment of A/Wild Bird/Taiwan/243/02(H3) and A/Mallard Duck/Taiwan/3.3/03 (H3). Conclusions: Residents in Kinmen islet frequently traveling from China need to monitor their flu A viruses that might be different from those obtained on the main Taiwan island.

The World Health Organization (WHO) Global Agenda on Influenza, published in the Weekly Epidemiological Report (WER) on 7 June 2002, was formulated by a consensus of influenza experts and identified strengthening national and international surveillance systems as a major priority. The benefits of improving surveillance include enhancing data available for vaccine strain selection and expanding the “early warning system” for the detection of variant strains of influenza, including those with pandemic potential. Evaluating the activities and physical facilities of the National Influenza Centers (NICs) was one of the key action steps identified in the WHO Global Agenda. An assessment of NICS was conducted using a survey tool developed by WHO. A synopsis of the preliminary results of this survey was published in the Weekly Epidemiological Report on 18 October 2002. A draft Action Plan for strengthening the global influenza surveillance system has been developed as a result of the assessment. A brief description of the assessment tool and key components of the draft Action Plan are outlined here.

Background: In February 2003, highly pathogenic avian influenza A H5N1 viruses reemerged in humans. Despite repeated outbreaks in domestic poultry in Hong Kong since 1999, this was the first isolation of H5N1 from humans since the outbreak in Hong Kong in 1997, which resulted in 18 human cases and 6 deaths. Methods: To better understand the antigenic relationship between the 2003 H5N1 human virus A/Hong Kong/213/03 (HK/213) and other H5 viruses, post- infection ferret sera or post-infection human sera were tested for reactivity by hemagglutination- inhibition and microneutralization assays with H5N1 viruses circulating in Hong Kong or elsewhere in Asia since 1997. Results: The H5N1 virus isolated from a 9-year-old male in Hong Kong was antigenically distinguishable from recent H5N1 viruses isolated from wild birds in Hong Kong and from the human 1997 H5N1 viruses, using post-infection ferret sera. Likewise, sera from this case patient, collected 22 days post-symptom onset, reacted to high titers with the homologous HK/213 virus, but gave eightfold lower titers with A/Hong Kong/156/97, and other H5 viruses. Conclusion: These results suggest that this recent human H5N1 virus is antigenically distinguishable from current and previously circulating H5N1 viruses from Asia, including the viruses previously isolated from humans. D 2004 Elsevier B.V. All rights reserved.

After nearly a decade's absence in most parts of the world, influenza B/Victoria/2/87-lineage (Vic) viruses reappeared and rapidly spread globally in 2001. Genetic analysis of the hemagglutinin (HA) and the neuraminidase (NA) genes revealed that the majority of Vic-lineage viruses isolated since 2001 possessed an NA similar to that of a B/Yamagata/16/88(Yam)-lineage reference strain. This finding indicated that reassortment of the NA gene between two lineages of influenza B viruses was associated with the global spread of Vic-lineage viruses. We also found that a small proportion of recent Vic-lineage viruses inherited both their HA and NA genes from a Vic-lineage parental virus isolated in 1997. Although HAs of these viruses were similar to that of the parental virus, four amino acid (AA) substitutions were identified in their NAs. Therefore, mutations detected in the NAs of those recent influenza B viruses also may have played a role in the global spread of Vic-lineage, non-reassortant viruses.

Background: Integrated virological surveillance systems along the transmission chains from animals to humans in areas with large swine/chicken/duck populations and numerous wild migrating birds grasps the frontline timing to detect novel flu viruses. Materials and methods: Animal and human virological surveillance systems have been established to monitor evolutional changes of flu viruses in the same and different hosts/geographical areas over years. Selected sentinel physicians collected specimens from influenza-like patients at local clinics/hospitals in: (a) counties with high populations of swine/avian/wild birds in Taiwan and (b) Kinmen islet—closest to China. Results: Novel animal and human influenza viruses (swine H1N2, avian H5 and H7 and human H3N2 within the lineage of A/Fujian/411/2002) were detected in 2002. However, subtypes of H5, H7 and H9 were not found in human isolates in Taiwan till Oct. 2003. Sentinel physician and school absenteeism surveillance systems were more sensitive than human laboratory and respiratory syndromic surveillance systems. Variation patterns of antigenic sites of HA1 in human versus animal flu A viruses were also quite different. Conclusion: The integrated surveillance is very useful in disease control and understanding conditions for the emergence of novel flu viruses with pandemic potential. D 2004 Elsevier B.V. All rights reserved.

Among the most challenging problems human geneticists currently face is how to move from the linkage mapping of genes for complex traits to the identification of genetic variation affecting those traits. In this manuscript, we review a variety of the analytic approaches most recently developed for the fine-mapping and positional cloning of genetic variation for complex phenotypes. These approaches, including multipoint linkage disequilibrium mapping, methods for explaining (in the statistical sense) the evidence for linkage, and combinatorial partitioning, can be quite computationally intensive. We also summarize recent research on rapid screening approaches, given that the number of polymorphisms routinely used in genetic studies is rapidly increasing.

Background: Avian influenza A H9N2 viruses are now widespread in poultry and were recently isolated from children with influenza-like illness in Hong Kong. The development and evaluation of vaccines against H9N2 viruses is a priority for pandemic preparedness. Methods: The immunogenicity and protective efficacy of live and inactivated H9N2 viruses that represented two antigenically and genetically distinct sublineages of Eurasian H9N2 viruses, G1 and G9, were evaluated in BALB/c mice. Mice were either infected intranasally with live virus or vaccinated intramuscularly with formalin-inactivated purified virus and challenged with G1-like and G9 viruses. Results: Infection or vaccination of mice with a G1-like virus induced substantially lower serum antibody responses against homologous virus, compared with mice infected or vaccinated with a G9 virus. The G1-like inactivated vaccine, but not the G9 vaccine, induced modest levels of antibody that cross-reacted with the heterologous H9 virus. Mice infected with either G1-like or G9 viruses or vaccinated with inactivated G1-like vaccine were protected from challenge with either virus. However, mice immunized with G9 vaccine, although completely protected from homologous challenge, were only partially protected against challenge with the G1-like virus. Conclusions: This study has identified differences in immunogenicity between the G1 and G9 H9N2 virus sublineages that have consequences for the design of optimal vaccines against circulating H9 viruses.

Background: During the 1999–2000 influenza season, influenza A (H3N2) viruses predominated in the United States and worldwide. Typically, influenza seasons in which influenza A (H3N2) viruses predominate are more severe than seasons in which influenza A (H1N1) and influenza B viruses are the primary circulating viruses. Three of the four indices used to monitor the impact of influenza in the US indicated that the 1999–2000 season was a typical H3N2 season; however, the P and I mortality reporting system and many media reports indicated that it was particularly severe. Methods: During the seasons covered (1996–1997 through 1999–2000), CDC received weekly reports from October through May from (1) collaborating laboratories that report on the total number of respiratory specimens tested and those that are influenza positive; (2) State and Territorial Epidemiologists who report on estimates of influenza activity in their state or territory; (3) sentinel physicians who report on the total number of patient visits and the number of cases of influenza-like illness (ILI); and (4) vital statistics offices in major US cities that report on the number of deaths related to pneumonia and influenza (P and I). Results: During the 1999–2000 influenza season, the percentage of respiratory specimens testing positive for influenza peaked at 33%; the state and territorial epidemiologists reports peaked at 44 states reporting either regional or widespread influenza activity; the percentage of patient visits for ILI peaked at 6%; and the proportion of deaths attributed to pneumonia and influenza peaked at 11.2%. Conclusions: Influenza activity during the 1999–2000 influenza season was similar to the three previous influenza seasons as indicated by reports from state and territorial epidemiologists, the percentage of respiratory specimens positive for influenza, and the proportion of visits for ILI. The percentage of deaths attributed to P and I reported by 122 US cities was higher than the peaks of the previous three seasons; however, part of the observed increase is apparently due to changes in reporting.

Background: Influenza surveillance in recent decades in Taiwan showed that the major virus isolates were A/Hong Kong/68-like (H3N2), A/England/42/72-like (H3N2), A/Taiwan/1/86 (H1N1), A/Texas/36/91-like (H1N1), A/Taiwan/112/96 (H1N1), and A/Taiwan/118/96 (H1N1) based on passive virologic surveillance before 1999. Materials and methods: To prevent the potential public health threat of a newly emerged influenza A strain like H5N1 in Hong Kong, seven surveillance systems have been established in Taiwan since 1999, including severe case reporting, sentinel physician, contract-laboratory, poultry market, domestic avian, wild bird and pig surveillance. Scientific exchanges and possible public health actions are discussed in regular meetings during the winter season. Results: The isolation rates of influenza A virus were higher among ducks than chickens in both domestic avian and market surveillance systems. Ten subtypes of influenza A virus can be found in migrating wild birds, which may serve as a reservoir for the genetic and antigenic changes of the virus. The H6 subtype was isolated from both wild birds and market surveillance systems. Pig surveillance found that H3 occurred more in the winter, whereas H1 appeared predominantly in the summer. Human virologic surveillance also demonstrated summer and winter peaks. Market surveillance in Taipei showed H6, H4, and H3 subtypes isolated from ducks only. Sentinel physician surveillance experience has taught us the importance of involving local clinics, particularly in farm areas. The traditional passive physician reporting surveillance provided limited information and it was replaced by severe case reporting. Most elderly physicians in Taiwan did not like to report influenza even after implementation of direct electronic reporting through a worldwide web system. More modern technology will be applied to increase the timeliness and representativeness of influenza surveillance in preparing for future pandemics. Conclusion: Integration of animal and human influenza surveillance systems in the high population density in Taiwan serves as a model in the efforts of global influenza surveillance to detect newly emerged influenza virus strains via several channels, particularly useful in detecting severity of cases and inter-species transmission.

Background: The hemagglutinins (HA) of human influenza viruses adapt during growth in chicken eggs. Amino acid replacements in HAs of egg-grown viruses generally appear on the terminal branches of phylogenetic trees. Our method for predicting human influenza HA evolution, which tracks replacements at positively selected codons across trees, is more accurate with terminal mutations excluded. Methods: We extracted the sequence data that contain the majority of egg-adapted replacements from our original data set, and replicated our prediction tests analyzing both internal and terminal mutations. We contrasted these results with those obtained analyzing (1) only internal mutations and (2) analyzing all mutations in our full data set. Results: Excluding egg-adapted replacements did not increase the accuracy of our predictions when other terminal mutations remained in the analysis. Deleting all terminal mutations produced the best prediction of success of viral lineages. Conclusions: Amino acid replacements on terminal branches interfered with our ability to identify which codons were under positive selection. Therefore, we recommend limiting such analyses to the internal branches of phylogenetic trees. The effects of terminal mutations may be caused by the criteria used for selecting viruses for HA sequencing.

Background : The accumulated wisdom is to update the vaccine strain to the expected epidemic strain only when there is at least a 4-fold difference [measured by the hemagglutination inhibition (HI) assay] between the current vaccine strain and the expected epidemic strain. In this study we investigate the effect, on repeat vaccinees, of updating the vaccine when there is a less than 4-fold difference. Methods : Using a computer model of the immune response to repeated vaccination, we simulated updating the vaccine on a 2-fold difference and compared this to not updating the vaccine, in each case predicting the vaccine efficacy in first-time and repeat vaccinees for a variety of possible epidemic strains. Results : Updating the vaccine strain on a 2-fold difference resulted in increased vaccine efficacy in repeat vaccinees compared to leaving the vaccine unchanged. Conclusions : These results suggest that updating the vaccine strain on a 2-fold difference between the existing vaccine strain and the expected epidemic strain will increase vaccine efficacy in repeat vaccinees compared to leaving the vaccine unchanged.

Background: Live influenza A vaccines (LIVs) for adults and children are prepared in Russia by reassorting current epidemic strains with cold-adapted (ca) attenuated donor viruses A/Leningrad/134/17/57 (H2N2) (Len/17) and A/Leningrad/134/47/57 (H2N2) (Len/47), respectively. Len/17 and Len/47 were derived from the A/Leningrad/134/57 (H2N2) wild-type virus after 17 and 47 passages in eggs at 25 °C. Two doses of Len/47-based vaccine have been used for vaccinating children. In a recent study, children were vaccinated with the Len/17-based LIV in order to determine if this vaccine is safe and immunogenic after a single dose vaccination. As part of this study, type A isolates obtained from children 3–6 years of age on the third day after vaccination were analyzed for genetic stability. Methods: PCR-restriction (RFLP) analysis was used to detect mutations in genomes of isolates. Results: Internal genes of Len/17 contain eight coding mutations in the PB2 (1), PB1 (2), PA (2), M1 (1), M2 (1) and NS (1) genes. All of these mutations, except one, were conserved in the genomes of all 28 strains studied. Similar to other studies with Len/17, the A-86-T change in the M2 protein, which was shown to be heterogeneous in the parent donor strain, was absent in 13 isolates. Since it was shown that mutations in polymerase genes play a leading role in the attenuation of Len/17, it is important to emphasize their conservation in all of the isolates. Moreover, an additional mutation in the PB2 protein (S-478-R), previously known only for the more attenuated Len/47 ca donor strain, was detected in 9 of 28 isolates, indicating that this mutation may also be heterogeneous in the donor virus. Conclusion: The Len/17-based LIV is genetically stable in children.

Introduction: This study examined alternative models for estimating influenza-attributable pneumonia and influenza (P&I) deaths in the United States which included influenza specific measures of viral activity as well as measures of RSV activity. Materials and methods: P&I deaths in the United States were modeled from 1976 to 1997. Predictors in the models included influenza subtypes A(H1N1), A(H3N2), and B and RSV. We analyzed data from 1976 to 1997. RSV data was available from 1990 through 1997 and therefore separate models were fit that included and excluded RSV measures. Results: Influenza accounted for an average of 4773 P&I deaths per year from 1976 to 1997. The results were highly correlated with previous estimates of P&I deaths in the United States (r=0.74, p

Background: Since Taiwan has high population density and a similar ecological environment to Mainland China, the epi-center of influenza viruses, it is very important to establish influenza virologic surveillance systems in both animal and human populations. The H5N1 Hong Kong Flu in 1997 and H9N2 in 1999 have showed that avian influenza viruses can cross the receptor of host species boundary and transmit to human. Therefore, the specific aim of this study is to understand the frequency of inter-species transmission in Taiwan. Methods: We have established an avian influenza virologic surveillance system in one of the largest live poultry markets in Taipei City from October 1999 to March 2000. Serum samples were collected from 341 blood donors, including veterinarians, poultry farm workers, and market employees. HI and microneutralization were used to detect specific antibody against H6, an endemic virus in chicken farms in southern Taiwan, and antibodies against H3, H4, and H9 viruses. Results: Among about 1300 fecal specimens of chickens and ducks collected, we isolated 12 H3 viruses, 14 H4 viruses, and 2 H6 viruses (i.e. 9 serotypes of HA and NA) from ducks. The isolation rates were 0% (0/580) and 7% (28/400) in chickens and ducks, respectively. Phylogenetic analysis of HA from 7 of our 12 H3 isolates showed the highest (93%) homology with A/equine/Jilin/89 (H3N8). Both phylogenetic relationship of HA and NP genes from selected representative strains (7 H3, 7 H4, and 2 H6) found they all fell into Eurasian lineage of avian influenza viruses. Their NP genes were away from the G1 lineage that was found in H5N1 strain isolated in Hong Kong in 1997. In addition, the results of HI and microneutralization tests found that they were all seronegative against two avian influenza virus strains [A/Duck/

Background: The A/Leningrad/134/17/57 (H2N2) (Len/17) cold-adapted (ca) master strain used as a donor of attenuation for preparing live attenuated influenza vaccine was derived from the A/Leningrad/134/57 wild-type virus (Len/wt) after 17 passages in eggs at 25 °C. In contrast to Len/wt, the Len/17 virus is sensitive to high (40 °C) temperature (ts), is able to grow at low (25 °C) temperature (ca), and is attenuated (att) for humans. Len/17 is different from Len/wt by eight coding mutations in internal genes PB2 (V-478-L), PB1 (K-265-N, V-591-I), PA (L-28-P, V-341-L), M1 (I-15-V), M2 (A-86-T) and NS2 (M-100-I). However, the role of individual mutant genes in the manifestation of the ts, ca, and att phenotypes was not established. To evaluate this, single gene reassortants (SGRs) with one mutated gene in the background of the Len/wt genome were studied. Methods: Since heterogeneity (A- or T-86 in the M2 gene) was noted in Len/17, the ts and ca clone 28 of this donor (Len/17-cl28) that has A-86 in M2 was used to prepare the SGRs. Preservation of mutations in SGRs was monitored by PCR-restriction (RFLP) analysis. Virus replication in ferret lungs was observed to evaluate the att phenotype of SGRs. Results: PB2- and PB1-SGRs each manifested the ts phenotype, and PB2-SGR demonstrated the ca phenotype as well. PA-SGR was ca, but not ts. Mutations in the M1 and NS2 genes did not affect the ts or ca phenotypes of corresponding SGRs. Ferrets infected with Len/17-cl28, Len/wt and PB2-, PB1- or PA-SGRs demonstrated comparable titers in nasal turbinates on the third day, while infectious virus was found only in the lungs of ferrets infected with Len/wt and PA-SGR. Conclusion: These data suggest that mutations in the PB2 (V-478-L) and PB1 (K-265-N, V-591-I) genes play a critical role in the attenuation of the ca Len/17 vaccine donor strain.

Background: Analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A and B viruses revealed worldwide circulation of multiple genetic lineages within the same type/subtype. Genetic reassortment between distinct genetic lineages was examined in this study. Methods: Nucleotide sequencing, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used. Phylogenetic studies were used to analyze sequence data. Results: Influenza A (H3N2) . The NA genes of H3N2 viruses were found to have evolved as three distinct lineages represented by A/Sydney/5/97, A/Panama/2007/99, and A/Moscow/10/99, respectively. Similar genetic groups were also found among the HA genes of these viruses. PCR/RFLP data of strains isolated between June 1999 and June 2000 showed that 122 of 141 viruses examined (87%) had their NA genes closely related to that of A/Moscow/10/99 virus; 14 of 141 viruses (10%) had A/Panama/2007/99-like NA genes, and only 5 strains (3%) had A/Sydney/5/97-like NA genes. Viruses bearing A/Panama/2007/99-like HA, and either A/Moscow/10/99-like, or A/Sydney/5/97-like NA were detected, indicating that genetic reassortments of the NA genes among different lineages of H3N2 viruses had occurred. Influenza A (H1N1) . Analyses showed that the NA and HA genes of the current strains each evolved as two genetically distinct lineages, represented by A/Bayern/7/95 and A/Beijing/262/95, respectively. The NA genes of the majority of circulating H1N1 viruses were closely related to that of the current vaccine strain A/New Caledonia/20/99 virus (A/Beijing/262/95 lineage). Influenza B . The NA genes of influenza B viruses, similar to their HA genes, were found to have diverged into two genetically and antigenically distinguishable lineages represented by B/Yamagata/16/88 (recently B/Yamanashi/166/98) and B/Victoria/2/87 (recently by B/Shandong/7/97) strains. Reassortants bearing the B/Yamagata-like HA and B/Victoria-like NA were isolated from several countries. Conclusion: Monitoring both HA and NA genes/antigens is important for appropriate vaccine strain selection.

In the United States, planning for the next influenza pandemic is occurring in parallel at national, state and local levels. Certain issues, such as conducting surveillance and purchasing pandemic vaccine, require coordination at the national level. However, most prevention and control actions will be implemented at the state and local level, which vary widely in terms of population demographics, culture (e.g., rural versus urban), and available resources. In 1995, a survey by the Council of State and Territorial Epidemiologists (CSTE) found that only 29 (59%) states perceived a need to develop a specific influenza pandemic plan for their jurisdiction. Since then, the process of developing state and local plans has gained considerable momentum. Integration of these efforts with the national planning process has been facilitated by: (1) the mutual involvement of state and federal staff in both processes; (2) the sharing of draft documents; (3) the ongoing occurrence of local and national coordinating meetings; and (4) the provision of financial resources by the federal government. So far, approximately 12 states either have drafted or begun drafting a state and local influenza pandemic plan. One of the benefits of the collaborative planning process has been the development of new working relationships and partnerships among several agencies at the state, local and national levels. Such efforts will improve our collective ability to rapidly investigate and control other emerging or re-emerging public health threats in the 21st century, be it a bioterrorist event, an influenza pandemic, or any other catastrophic health event.