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Influenza surveillance of some surveillance sites in Fujian Province, China from January 2001 to December 2002
International Congress Series 1263 (2004) 255 – 258
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Influenza surveillance of some surveillance sites in Fujian Province, China from January 2001 to December 2002 Wen-Qiong Xiu a,*, Shi-Qin Yang a, Shuang-Feng Lv a, Qing-Zhen Tang b, Hui-Qin Chen b, Xiao-Na Shen a a
Department of Viral Disease, Fujian Centers for Disease Control and Prevention, Jintai Road 76, Fuzhou, Fujian 350001, PR China b Fujian Provincial Maternity and Child Health Hospital, Fuzhou, Fujian, PR China
Abstract. In order to provide control and prevention measures against an influenza pandemic, we investigated the epidemic characteristics of influenza from January 2001 in some surveillance sites of Fujian province, especially in Fuzhou city. We isolated 42 influenza viruses in 2001, 32 of them were H1N1, 10 isolates belonged to the B/Yamagata lineage. We did not isolate H3N2 in 2001. But in 2002, we isolated 44 influenza viruses; 22 of them were H3N2, only one isolate was H1N1, the other 21 isolates were B type. Five of the B type were B/Yamagata and 16 were B/Victoria. Eight isolates of B/Victoria were isolated from a primary school in Fuzhou in December 2002 for an influenza outbreak. In Fuzhou, the epidemic was predominated by H1N1 in 2001. But in 2002, the epidemic was predominated by H3N2 and B/Victoria. There were two epidemic peaks in 2001 and 2002, but the peaks’ time and predominant strains were different. It is so strange that we isolated 32 isolates of the H1N1 influenza virus with embryonated chicken eggs in 2001, but in 2002 we only isolated 1 isolate of H1N1 with embryonated chicken eggs. In 2002, most influenza viruses, especially H3N2, were isolated by MDCK cells. The epidemic strain of the influenza virus changed all the time, and the influenza virus also changed in its way. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza virus; Surveillance
1. Introduction To date, vaccines do not yet control influenza and the epidemic of influenza occurs every year. It still causes significant morbidity and mortality worldwide. It is well known that the epidemic of influenza often occurred in China. It is possible that China was the birthplace of influenza epidemic strains. Influenza viruses are also circulating continuously
* Corresponding author. Tel.: +86-591-7519604; fax: +86-591-7533291. E-mail address: [email protected] (W.-Q. Xiu). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.123
256
W.-Q. Xiu et al. / International Congress Series 1263 (2004) 255–258
in Fujian. We devoted ourselves to the study of epidemic characteristics of influenza viruses isolated in Fujian over the years. 2. Materials and methods 2.1. Collection of clinical specimens We selected two hospitals in Fuzhou as sentinel surveillance locations for influenza-like specimen collection and epidemiology surveillance. Most cases of influenza-like patients appeared to have febrile common colds, fever z 38 jC. Other typical features of influenza included fever, headache, muscle aches, cough and sore throat. The patient specimens were collected with throat swabs. To maximize the probability of isolating the virus, samples should be taken within 72 h of onset of illness. We collected the specimens in Eagle’s MEM at hypothermia, transported them to the laboratory as soon as possible and stored them at 4 jC. We inoculated the specimens on MDCK cells or embryonated chicken eggs within 48 h. Then the specimens were stored at 70 jC. 2.2. Materials and reagents Embryonated chicken eggs were purchased from Fujian Biological and Pharmaceutical Factory. MDCK cells and reference antisera for the identification of the influenza virus were provided by the National Influenza Center, Beijing, China. Eagle’s MEM was commonly used as the transport medium for collecting specimens. The addition of antibiotics (2000 units/ml penicillin G, and 2000 Ag/ml streptomycin) and 2 Ag/ml antimycotics helped prevent microbial growth. MDCK cells were grown in Eagle’s MEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ ml penicillin G, and 100 Ag/ml streptomycin. Eagle’s MEM without bovine serum can be used as washing fluid. Maintaining fluids contained 2– 3 Ag/ml trypsin. The pH of all fluids was 7.6. 2.3. Virus isolation 2.3.1. Inoculation of embryonated chicken eggs [1] Embryonated chicken eggs were about 9 days old. Three eggs per specimen were usually inoculated. We usually inoculated 100 Al of the specimen into the amniotic cavity and 100 Al into the allantoic cavity. We incubated the eggs at 35 jC for 72 h. Eggs were chilled at + 4 jC overnight before harvesting. We performed a hemagglutination (HA) test with 1% guinea pig RBCS. If no HA was present, we passaged the specimen one more time. 2.3.2. Inoculation of cell culture [1] When obtaining a confluent monolayer of MDCK cells in a flask, we decanted the growth medium and washed two times with washing fluids. We inoculated 500– 1000 Al of each specimen into a flask and allowed the inoculum to adsorb for 1– 2 h at 35 jC. We decanted infected fluids, washed one time with washing fluids and added 3 ml of maintaining fluids to each flask. The cells were incubated at 35 jC and observed daily
W.-Q. Xiu et al. / International Congress Series 1263 (2004) 255–258
257
for cytopathogenic effect (CPE). We harvested the cell culture if 3+ or 4+ CPE was observed by collecting supernatant fluids and harvested them by day 6 or 7, even if no CPE was observed. Cells were chilled at 20 jC before harvesting. We performed an HA test and stored at 4 jC. 2.4. Virus identification [1] We identified influenza virus by hemagglutination inhibition (HAI) test with reference antisera for the identification of the influenza virus. Then we stored the isolates at 70 jC within 2 days of harvest. We sent the isolates to the National Influenza Center of China within 10 days for final identification. 3. Results The total number of specimens tested was 543 in 2001 [2] and 804 in 2002. We isolated 42 influenza viruses in 2001, 32 of them were H1N1 and 10 isolates belonged to B/Yamagata lineage. We did not isolate H3N2 in 2001. The positive rate of isolation was 7.7%. In 2001 (Fig. 1), we did not isolate the influenza virus in January, February, May and June. In other months, the number of positive/number of specimens tested were: March 6/48 (B type); April 2/57 (B type); July 5/87 (4 were H1N1, 1 was B type); August 19/130 (18 were H1N1, 1 was B type); September 3/34 (H1N1); October 3/34 (H1N1); November 1/22 (H1N1); December 3/51 (H1N1). In 2002, we isolated 44 influenza viruses, 22 of them were H3N2, only one isolate was H1N1, the other 21 isolates were B type. Five of the B type were B/Yamagata lineage and 16 were B/ Victoria lineage. Eight isolates of B/Victoria were isolated from a primary school in Fuzhou in December 2002 for influenza outbreak. In 2002 (Fig. 1), the positive rate of isolation was 5.5%. We did not isolate the influenza virus in February, April and September. In other months, the number of positive/number of specimens tested were: January 2/40 (H3N2); March 1/37 (H3N2); May 2/77 (1 was H1N1, 1 was H3N2); June 3/62 (H3N2); July 13/140 (H3N2); August 2/96 (H3N2); October 5/83 (B/ Yamagata); November 1/54 (B/Victoria); and December 15/67 (B/Victoria). There were two epidemic peaks in 2001 (Fig. 2). The small epidemic peak was in spring (from March to April) and B type (B/Yamagata) was the predominated strain; the large epidemic peak was in summer (from July to September), and H1N1 was the predominated strain. There were also two epidemic peaks in 2002 (Fig. 2), but the
Fig. 1. Number of influenza virus isolated per month in Fujian, China in 2001 and 2002.
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Fig. 2. Isolation rates of influenza virus of Fujian, China in 2001 and 2002.
peak time changed. One peak was in summer (from June to August) and H3N2 was the predominated strain. The other was in winter (after October) and B/Victoria was the predominated strain. 4. Discussion It is so strange that we isolated 32 isolates of H1N1 influenza virus with embryonated chicken eggs in 2001, but in 2002 we only isolated 1 isolate of H1N1 with embryonated chicken eggs. In 2002, most influenza viruses, especially H3N2 were isolated by MDCK cells. MDCK cells were more sensitive to the H3N2 influenza virus. It seems influenza viruses easily underwent constant antigenic change. Within countries or regions, the predominant circulating virus type or subtype varied and changed frequently as the season progressed. References [1] Y.J. Guo, X.W. Cheng, Influenza Viruses and Experimental Techniques, China Sanxia Publishing Company, Beijing, 1997. [2] W.Q. Xiu, et al., Influenza surveillance of some surveillance sites in Fujian province in 2001, Strait Journal of Preventive Medicine 9 (3) (2003) 50 – 51.
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Influenza surveillance of some surveillance sites in Fujian Province, China from January 2001 to December 2002 Wen-Qiong Xiu a,*, Shi-Qin Yang a, Shuang-Feng Lv a, Qing-Zhen Tang b, Hui-Qin Chen b, Xiao-Na Shen a a
Department of Viral Disease, Fujian Centers for Disease Control and Prevention, Jintai Road 76, Fuzhou, Fujian 350001, PR China b Fujian Provincial Maternity and Child Health Hospital, Fuzhou, Fujian, PR China
Abstract. In order to provide control and prevention measures against an influenza pandemic, we investigated the epidemic characteristics of influenza from January 2001 in some surveillance sites of Fujian province, especially in Fuzhou city. We isolated 42 influenza viruses in 2001, 32 of them were H1N1, 10 isolates belonged to the B/Yamagata lineage. We did not isolate H3N2 in 2001. But in 2002, we isolated 44 influenza viruses; 22 of them were H3N2, only one isolate was H1N1, the other 21 isolates were B type. Five of the B type were B/Yamagata and 16 were B/Victoria. Eight isolates of B/Victoria were isolated from a primary school in Fuzhou in December 2002 for an influenza outbreak. In Fuzhou, the epidemic was predominated by H1N1 in 2001. But in 2002, the epidemic was predominated by H3N2 and B/Victoria. There were two epidemic peaks in 2001 and 2002, but the peaks’ time and predominant strains were different. It is so strange that we isolated 32 isolates of the H1N1 influenza virus with embryonated chicken eggs in 2001, but in 2002 we only isolated 1 isolate of H1N1 with embryonated chicken eggs. In 2002, most influenza viruses, especially H3N2, were isolated by MDCK cells. The epidemic strain of the influenza virus changed all the time, and the influenza virus also changed in its way. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza virus; Surveillance
1. Introduction To date, vaccines do not yet control influenza and the epidemic of influenza occurs every year. It still causes significant morbidity and mortality worldwide. It is well known that the epidemic of influenza often occurred in China. It is possible that China was the birthplace of influenza epidemic strains. Influenza viruses are also circulating continuously
* Corresponding author. Tel.: +86-591-7519604; fax: +86-591-7533291. E-mail address: [email protected] (W.-Q. Xiu). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.123
256
W.-Q. Xiu et al. / International Congress Series 1263 (2004) 255–258
in Fujian. We devoted ourselves to the study of epidemic characteristics of influenza viruses isolated in Fujian over the years. 2. Materials and methods 2.1. Collection of clinical specimens We selected two hospitals in Fuzhou as sentinel surveillance locations for influenza-like specimen collection and epidemiology surveillance. Most cases of influenza-like patients appeared to have febrile common colds, fever z 38 jC. Other typical features of influenza included fever, headache, muscle aches, cough and sore throat. The patient specimens were collected with throat swabs. To maximize the probability of isolating the virus, samples should be taken within 72 h of onset of illness. We collected the specimens in Eagle’s MEM at hypothermia, transported them to the laboratory as soon as possible and stored them at 4 jC. We inoculated the specimens on MDCK cells or embryonated chicken eggs within 48 h. Then the specimens were stored at 70 jC. 2.2. Materials and reagents Embryonated chicken eggs were purchased from Fujian Biological and Pharmaceutical Factory. MDCK cells and reference antisera for the identification of the influenza virus were provided by the National Influenza Center, Beijing, China. Eagle’s MEM was commonly used as the transport medium for collecting specimens. The addition of antibiotics (2000 units/ml penicillin G, and 2000 Ag/ml streptomycin) and 2 Ag/ml antimycotics helped prevent microbial growth. MDCK cells were grown in Eagle’s MEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ ml penicillin G, and 100 Ag/ml streptomycin. Eagle’s MEM without bovine serum can be used as washing fluid. Maintaining fluids contained 2– 3 Ag/ml trypsin. The pH of all fluids was 7.6. 2.3. Virus isolation 2.3.1. Inoculation of embryonated chicken eggs [1] Embryonated chicken eggs were about 9 days old. Three eggs per specimen were usually inoculated. We usually inoculated 100 Al of the specimen into the amniotic cavity and 100 Al into the allantoic cavity. We incubated the eggs at 35 jC for 72 h. Eggs were chilled at + 4 jC overnight before harvesting. We performed a hemagglutination (HA) test with 1% guinea pig RBCS. If no HA was present, we passaged the specimen one more time. 2.3.2. Inoculation of cell culture [1] When obtaining a confluent monolayer of MDCK cells in a flask, we decanted the growth medium and washed two times with washing fluids. We inoculated 500– 1000 Al of each specimen into a flask and allowed the inoculum to adsorb for 1– 2 h at 35 jC. We decanted infected fluids, washed one time with washing fluids and added 3 ml of maintaining fluids to each flask. The cells were incubated at 35 jC and observed daily
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257
for cytopathogenic effect (CPE). We harvested the cell culture if 3+ or 4+ CPE was observed by collecting supernatant fluids and harvested them by day 6 or 7, even if no CPE was observed. Cells were chilled at 20 jC before harvesting. We performed an HA test and stored at 4 jC. 2.4. Virus identification [1] We identified influenza virus by hemagglutination inhibition (HAI) test with reference antisera for the identification of the influenza virus. Then we stored the isolates at 70 jC within 2 days of harvest. We sent the isolates to the National Influenza Center of China within 10 days for final identification. 3. Results The total number of specimens tested was 543 in 2001 [2] and 804 in 2002. We isolated 42 influenza viruses in 2001, 32 of them were H1N1 and 10 isolates belonged to B/Yamagata lineage. We did not isolate H3N2 in 2001. The positive rate of isolation was 7.7%. In 2001 (Fig. 1), we did not isolate the influenza virus in January, February, May and June. In other months, the number of positive/number of specimens tested were: March 6/48 (B type); April 2/57 (B type); July 5/87 (4 were H1N1, 1 was B type); August 19/130 (18 were H1N1, 1 was B type); September 3/34 (H1N1); October 3/34 (H1N1); November 1/22 (H1N1); December 3/51 (H1N1). In 2002, we isolated 44 influenza viruses, 22 of them were H3N2, only one isolate was H1N1, the other 21 isolates were B type. Five of the B type were B/Yamagata lineage and 16 were B/ Victoria lineage. Eight isolates of B/Victoria were isolated from a primary school in Fuzhou in December 2002 for influenza outbreak. In 2002 (Fig. 1), the positive rate of isolation was 5.5%. We did not isolate the influenza virus in February, April and September. In other months, the number of positive/number of specimens tested were: January 2/40 (H3N2); March 1/37 (H3N2); May 2/77 (1 was H1N1, 1 was H3N2); June 3/62 (H3N2); July 13/140 (H3N2); August 2/96 (H3N2); October 5/83 (B/ Yamagata); November 1/54 (B/Victoria); and December 15/67 (B/Victoria). There were two epidemic peaks in 2001 (Fig. 2). The small epidemic peak was in spring (from March to April) and B type (B/Yamagata) was the predominated strain; the large epidemic peak was in summer (from July to September), and H1N1 was the predominated strain. There were also two epidemic peaks in 2002 (Fig. 2), but the
Fig. 1. Number of influenza virus isolated per month in Fujian, China in 2001 and 2002.
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Fig. 2. Isolation rates of influenza virus of Fujian, China in 2001 and 2002.
peak time changed. One peak was in summer (from June to August) and H3N2 was the predominated strain. The other was in winter (after October) and B/Victoria was the predominated strain. 4. Discussion It is so strange that we isolated 32 isolates of H1N1 influenza virus with embryonated chicken eggs in 2001, but in 2002 we only isolated 1 isolate of H1N1 with embryonated chicken eggs. In 2002, most influenza viruses, especially H3N2 were isolated by MDCK cells. MDCK cells were more sensitive to the H3N2 influenza virus. It seems influenza viruses easily underwent constant antigenic change. Within countries or regions, the predominant circulating virus type or subtype varied and changed frequently as the season progressed. References [1] Y.J. Guo, X.W. Cheng, Influenza Viruses and Experimental Techniques, China Sanxia Publishing Company, Beijing, 1997. [2] W.Q. Xiu, et al., Influenza surveillance of some surveillance sites in Fujian province in 2001, Strait Journal of Preventive Medicine 9 (3) (2003) 50 – 51.